Uptake Of Phospholipids Research Articles

Targeting the SREBP-1/Hsa-Mir-497/SCAP/FASN Oncometabolic Axis Inhibits the Cancer Stem-like and Chemoresistant Phenotype of Non-Small Cell Lung Carcinoma Cells.

Background: Lung cancer remains a leading cause of cancer-related death, with an annual global mortality rate of 18.4%. Despite advances in diagnostic and therapeutic technologies, non–small cell lung carcinoma (NSCLC) continues to be characterized by a poor prognosis. This may be associated with the enrichment of cancer stem cells (CSCs) and the development of chemoresistance—a double-edged challenge that continues to impede the improvement of long-term outcomes. Metabolic reprogramming is a new hallmark of cancer. Sterol regulatory element-binding proteins (SREBPs) play crucial regulatory roles in the synthesis and uptake of cholesterol, fatty acids, and phospholipids. Recent evidence has demonstrated that SREBP-1 is upregulated in several cancer types. However, its role in lung cancer remains unclear. Objective: This study investigated the role of SREBP-1 in NSCLC biology, progression, and therapeutic response and explored the therapeutic exploitability of SREBP-1 and SREBP-1-dependent oncometabolic signaling and miRNA epigenetic regulation. Methods: We analyzed SREBP-1 levels and biological functions in clinical samples and the human NSCLC cell lines H441 and A549 through shRNA-based knock down of SREBP function, cisplatin-resistant clone generation, immunohistochemical staining of clinical samples, and cell viability, sphere-formation, Western blot, and quantitative PCR assays. We conducted in-silico analysis of miRNA expression in NSCLC samples by using the Gene Expression Omnibus (GSE102286) database. Results: We demonstrated that SREBP-1 and SCAP are highly expressed in NSCLC and are positively correlated with the aggressive phenotypes of NSCLC cells. In addition, downregulation of the expression of tumor-suppressing hsa-miR-497-5p, which predictively targets SREBP-1, was observed. We also demonstrated that SREBP-1/SCAP/FASN lipogenic signaling plays a key role in CSCs-like and chemoresistant NSCLC phenotypes, especially because the fatostatin or shRNA targeting of SREBP-1 significantly suppressed the viability, cisplatin resistance, and cancer stemness of NSCLC cells and because treatment induced the expression of hsa-miR-497. Conclusion: Targeting the SREBP-1/hsa-miR-497 signaling axis is a potentially effective anticancer therapeutic strategy for NSCLC.

Digestion and absorption of dietary glycerophospholipids in the small intestine: Their significance as carrier molecules of choline and n-3 polyunsaturated fatty acids

Phospholipid is an important constituent of the body, as the main component of cell membrane and various lipoproteins . As dietary lipids, the normal dietary intake of phospholipids is estimated to be about 1–10% of the total daily fat intake , with triacylglycerol being the main compound. Phospholipid intake, especially phosphatidylcholine , is important as they supply choline and essential fatty acids , which are essential for good health and involved in various cellular functions. Recently, phospholipid uptake in the enterocyte has been found to be a key step in intestinal dietary lipid absorption . In this review, the general concept of phospholipid digestion, absorption, and metabolism, especially concerning phosphatidylcholine, is summarized. In addition, the nutritional role of phospholipids as carrier molecules of choline and n-3 polyunsaturated fatty acids has been discussed. • Dietary phospholipids are mainly composed of phosphatidylcholine. • Phosphatidylcholine is a good dietary source of choline. • Dietary phospholipids also act as a carrier molecule of essential fatty acids. • The beneficial health effects of dietary phospholipids have been widely investigated. • The general concept of phospholipid digestion and absorption is summarized.

Milk phospholipid antioxidant activity and digestibility: Kinetics of fatty acids and choline release

Abstract Milk phospholipids have been used as functional ingredients in foods. However, there have been no reports on how the polarity of lipids affects their digestibility. This study aims to isolate milk phospholipids from dairy products, to characterize them chromatographically, and assess their digestibility and antioxidant activities in vitro. The results revealed that their lipolysis reaction rate constants were significantly different ( p 0.05 ) between milk phospholipids and triacylglycerols, and thus the degradation of both lipids complied with first-order reaction kinetics. Furthermore, the cellular uptake of milk phospholipids was determined with HT-29 cell model, and they were not found to be absorbed intact during intestinal digestion. Milk phospholipids exhibited significant antioxidant activity in vitro, while their cellular antioxidant activity was very limited. The results of this study provide useful information for the design of milk phospholipid-based carrier systems.

Endocytic recycling pathways and the regulatory mechanisms

Endocytic transport is imperative for the exchange of information between cells and the external environment. Specifically, the process of endocytic transport comprises precise regulation of uptake and sorting of extracellular macromolecules, phospholipids, and membrane proteins. In the endocytic transport system, the recycling pathways are responsible for delivering membrane proteins and phospholipids back to the plasma membrane. Thus, endocytic recycling plays critical roles in various biological processes, including nutrient absorption, cell polarity establishment, cell migration, cell division, synaptic plasticity, immune response, and growth factor receptor regulation. There are two essential types of recycling pathways in eukaryotic cells, recycling of clathrin-dependent endocytic cargos (CDE recycling) and recycling of clathrin-independent endocytic cargos (CIE recycling). The transferrin receptor TfR and the low-density lipoprotein receptor LDLR, which have essential physiological roles in vivo, are representative membrane proteins of the CDE recycling transport. In recent years, various membrane proteins governed by CIE recycling transport have been identified, including IL2 receptor α-subunit, major histocompatibility complex MHC Class I, and glucose transporter GLUT4. Therefore, the investigation of the regulatory mechanisms of CIE recycling has drawn notable attention in the field. Moreover, CIE recycling research presents fundamental significance in cell biology, which also provides scientific evidence and potential therapeutic clues for the diagnosis and treatment strategies of diseases such as type 2 diabetes and cancer. Compared with the CDE recycling, the study on CIE recycling started later, and there is much to be learned of its regulatory mechanisms. To this end, this review summarizes the features of endocytic recycling pathways, focuses on the molecular basis of CIE recycling regulation and elaborates on the latest progress and newly developed research model systems in the field of CIE recycling.

Photostability of quantum dot micelles under ultraviolet irradiation.

Phospholipid quantum dot micelles are useful for bio-applications because of their amphiphilicity and exceptional biocompatibilities. We investigated the uptake of phospholipid [polyethylene glycol (PEG), biotin, and folic acid terminated] modified CdSe/ZnS quantum dot micelles by cancer cells and its photostability under ultrviolet light in the C spectrum (UV-C) (254nm) or UV-A (365nm) light irradiation. The stability of micelles to the exposure of UV-C and UV-A light was assessed. Biotin-modified quantum dot micelles give photoluminescence enhancement under UV-C light irradiation. Folate modified micelle under UV-C and UV-A results show considerable photoluminescence enhancement. Photoluminescence lifetime measurements showed 7.04, 8.11 and 11.42ns for PEG, folate, and biotin terminated phospholipid micelles, respectively. Folate and biotin-modified quantum dot micelles showed excellent uptake by HeLa cells under fluorescence confocal microscopy. Phospholipid CdSe/ZnS quantum dot micelles can be potentially used for diagnosis and treatment of cancer in the future.

Expression of miR-33 from an SREBP2 intron inhibits the expression of the fatty acid oxidation-regulatory genes CROT and HADHB in chicken liver

ABSTRACT1. Limiting the growth of adipose tissue in chickens is a major issue in the poultry industry. In chickens, de novo synthesis of lipids occurs primarily in the liver. Thus, it is necessary to understand how fatty acid accumulation in the liver is controlled. The miR-33 is an intronic microRNA (miRNA) of the chicken sterol regulatory element binding transcription factor 2 (SREBF2), which is a master switch in activating many genes involved in the uptake and synthesis of cholesterol, triglycerides, fatty acids and phospholipids.2. In the current study, the genes CROT and HADHB known to encode enzymes critical for fatty acid oxidation were predicted to be potential targets of miR-33 in chickens via the miRNA target prediction programs ‘miRanda’ and ‘TargetScan’. Co-transfection and dual-luciferase reporter assays showed that the expression of luciferase reporter gene linked to the 3ʹ-untranslated region (3ʹUTR) of the chicken CROT and HADHB mRNA was down-regulated by overexpression of the chicken miR-33 (P < 0.05). This down-regulation was completely abolished when the predicted miR-33 target sites in the CROT and HADHB 3ʹUTR were mutated.3. Transfecting miR-33 mimics into the LMH cells led to a decrease in the mRNA expression of CROT and HADHB (P < 0.01), and this transfection had a similar effect on the proteins (P < 0.05). In contrast, the expression of CROT in primary chicken hepatocytes was up-regulated after transfection with the miR-33 inhibitor LNA-anti-miR-33 (P < 0.05).4. Using quantitative RT-PCR, it was shown that the expression of miR-33 was increased in the chicken liver from day 0 to day 49 of age, whereas the CROT and HADHB mRNA levels decreased during the same period.5. These findings support the conclusion that miR-33 might play an important role in lipid metabolism in the chicken liver by negatively regulating the expression of the CROT and HADHB genes, which encode enzymes critical for lipid oxidation.

CD36 in chronic kidney disease: novel insights and therapeutic opportunities.

CD36 (also known as scavenger receptor B2) is a multifunctional receptor that mediates the binding and cellular uptake of long-chain fatty acids, oxidized lipids and phospholipids, advanced oxidation protein products, thrombospondin and advanced glycation end products, and has roles in lipid accumulation, inflammatory signalling, energy reprogramming, apoptosis and kidney fibrosis. Renal CD36 is mainly expressed in tubular epithelial cells, podocytes and mesangial cells, and is markedly upregulated in the setting of chronic kidney disease (CKD). As fatty acids are the preferred energy source for proximal tubule cells, a reduction in fatty acid oxidation in CKD affects kidney lipid metabolism by disrupting the balance between fatty acid synthesis, uptake and consumption. The outcome is intracellular lipid accumulation, which has an important role in the pathogenesis of kidney fibrosis. In experimental models, antagonist blockade or genetic knockout of CD36 prevents kidney injury, suggesting that CD36 could be a novel target for therapy. Here, we discuss the regulation and post-translational modification of CD36, its role in renal pathophysiology and its potential as a biomarker and as a therapeutic target for the prevention of kidney fibrosis.

A phospholipid uptake system in the model plant Arabidopsis thaliana.

Plants use solar energy to produce lipids directly from inorganic elements and are not thought to require molecular systems for lipid uptake from the environment. Here we show that Arabidopsis thaliana Aminophospholipid ATPase10 (ALA10) is a P4-type ATPase flippase that internalizes exogenous phospholipids across the plasma membrane, after which they are rapidly metabolized. ALA10 expression and phospholipid uptake are high in the epidermal cells of the root tip and in guard cells, the latter of which regulate the size of stomatal apertures to modulate gas exchange. ALA10-knockout mutants exhibit reduced phospholipid uptake at the root tips and guard cells and are affected in growth and transpiration. The presence of a phospholipid uptake system in plants is surprising. Our results suggest that one possible physiological role of this system is to internalize lysophosphatidylcholine, a signalling lipid involved in root development and stomatal control.

Ras‐Induced Macropinocytosis: Metabolic adaptation to signaling in the absence of adequate nutrients

Under conditions of an abundant supply of extracellular glucose and glutamine, cancer cells synthesize lipids, nucleic acids, and non‐essential amino acids from these precursors. However, when cancer grows to exceed its vascular supply, cells must adapt to lower availability of essential nutrients and oxygen. In the past year, we have seen a major breakthrough in the discovery of novel mechanisms by which cancer cells adapt to metabolic stress. It was previously determined that glutamine‐dependent lipid synthesis could support de novo lipid synthesis under hypoxia but metabolic tracing studies uncovered that over 50% of lipids used in phospholipid biosynthesis in hypoxic cells come from exogenous sources. We were able to determine that hypoxia‐induced macropinocytosis facilitates the uptake and incorporation of unsaturated phospholipids to maintain effective unsaturated lipid levels under conditions in which stearoyl‐CoA desaturase is inhibited by oxygen limitation. Follow up studies have demonstrated that this property of cellular scavenging of extracellular macromolecules is induced in a Ras‐dependent fashion in transformed cells and provides a potent mechanism by which Ras‐transformed cells can adapt to metabolic stress in their environment. This pathway of cellular acquisition of bioenergetic substrates has previously escaped detection because, in complete medium, the utilization of macropinocytosed proteins as metabolic substrates is actively suppressed. The molecular details of this suppression and the implication of these studies to understanding both cancer and wound repair will be discussed.

Analytical characterization of the role of phospholipids in platelet adhesion and secretion.

The cellular phospholipid membrane plays an important role in cell function and cell–cell communication, but its biocomplexity and dynamic nature presents a challenge for examining cellular uptake of phospholipids and the resultant effects on cell function. Platelets, small anuclear circulating cell bodies that influence a wide variety of physiological functions through their dynamic secretory and adhesion behavior, present an ideal platform for exploring the effects of exogenous phospholipids on membrane phospholipid content and cell function. In this work, a broad range of platelet functions are quantitatively assessed by leveraging a variety of analytical chemistry techniques, including ultraperformance liquid chromatography–tandem electrospray ionization mass spectrometry (UPLC–MS/MS), vasculature-mimicking microfluidic analysis, and single cell carbon-fiber microelectrode amperometry (CFMA). The relative enrichments of phosphatidylserine (PS) and phosphatidylethanolamine (PE) were characterized with UPLC–MS/MS, and the effects of the enrichment of these two phospholipids on both platelet secretory behavior and adhesion were examined. Results show that, in fact, both PS and PE influence platelet adhesion and secretion. PS was enriched dramatically and decreased platelet adhesion as well as secretion from δ-, α-, and lysosomal granules. PE enrichment was moderate and increased secretion from platelet lysosomes. These insights illuminate the critical connection between membrane phospholipid character and platelet behavior, and both the methods and results presented herein are likely translatable to other mammalian cell systems.

Expression of miR-33 from an SREBF2 Intron Targets the FTO Gene in the Chicken

The sterol regulatory element binding transcription factor 2 (SREBF2) gene encodes a transcription factor that activates the expression of many genes involved in the synthesis and uptake of cholesterol, fatty acids, triglycerides, and phospholipids. Through bioinformatics, we found that intron 16 of the chicken SREBF2 gene might encode the chicken miR-33. Using quantitative RT-PCR, we detected the expression of miR-33 in a variety of chicken tissues including skeletal muscle, adipose tissue, and liver. Three hundred and seventy eight genes were predicted to be potential targets of miR-33 in chickens via miRNA target prediction programs “miRanda” and “TargetScan”. Among these targets, the gene FTO (fat mass and obesity associated) encodes a Fe(II)- and 2-oxoglutarate-dependent nucleic acid demethylase that regulates lipid metabolism, and the possibility that its expression is negatively regulated by miR-33 in the chicken liver was therefore further studied. Co-transfection and dual-luciferase reporter assays showed that the expression of luciferase reporter gene linked to the 3′-untranslated region (3′UTR) of the chicken FTO mRNA was down-regulated by overexpression of the chicken miR-33 in the C2C12 cells (P<0.05). Furthermore, this down-regulation was completely abolished when the predicted miR-33 target site in the FTO 3′UTR was mutated. In contrast, the expression of FTO mRNA in the primary chicken hepatocytes was up-regulated after transfection with the miR-33 inhibitor LNA-anti-miR-33. Using quantitative RT-PCR, we also found that the expression of miR-33 was increased in the chicken liver from day 0 to day 49 of age, whereas that of the FTO mRNA was decreased during the same age period. These data together suggest that miR-33 might play an important role in lipid metabolism in the chicken liver by negatively regulating the expression of the FTO gene.

Abstract PL02-03: Ras-induced macropinocytosis: Metabolic adaptation to signaling in the absence of adequate nutrients.

Abstract Under conditions of an abundant supply of extracellular glucose and glutamine, cancer cells synthesize lipids, nucleic acids, and non-essential amino acids from these precursors. However, when cancer grows to exceed its vascular supply, cells must adapt to lower availability of essential nutrients and oxygen. This past year we have seen a significant breakthrough in the discovery of novel mechanisms by which cancer cells adapt to metabolic stress. It was previously determined that glutamine-dependent lipid synthesis could support de novo lipid synthesis under hypoxia but metabolic tracing studies uncovered that over 50% of lipids used in phospholipid biosynthesis in hypoxic cells come from exogenous sources. We were able to determine that hypoxia-induced macropinocytosis facilitates the uptake and incorporation of unsaturated phospholipids to maintain effective unsaturated lipid levels under conditions in which stearoyl-CoA desaturase is inhibited by oxygen limitation. Follow up studies, in collaboration with others, have demonstrated that this property of cellular scavenging of extracellular macromolecules is induced in a Ras-dependent fashion in transformed cells and provides a potent mechanism by which Ras-transformed cells can adapt to metabolic stress in their environment. How these insights can be used to develop novel therapeutic strategies to address Ras-induced tumors will be discussed. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):PL02-03. Citation Format: Craig B. Thompson. Ras-induced macropinocytosis: Metabolic adaptation to signaling in the absence of adequate nutrients. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr PL02-03.

Membrane lipid co-aggregation with α-synuclein fibrils.

Amyloid deposits from several human diseases have been found to contain membrane lipids. Co-aggregation of lipids and amyloid proteins in amyloid aggregates, and the related extraction of lipids from cellular membranes, can influence structure and function in both the membrane and the formed amyloid deposit. Co-aggregation can therefore have important implications for the pathological consequences of amyloid formation. Still, very little is known about the mechanism behind co-aggregation and molecular structure in the formed aggregates. To address this, we study in vitro co-aggregation by incubating phospholipid model membranes with the Parkinson’s disease-associated protein, α-synuclein, in monomeric form. After aggregation, we find spontaneous uptake of phospholipids from anionic model membranes into the amyloid fibrils. Phospholipid quantification, polarization transfer solid-state NMR and cryo-TEM together reveal co-aggregation of phospholipids and α-synuclein in a saturable manner with a strong dependence on lipid composition. At low lipid to protein ratios, there is a close association of phospholipids to the fibril structure, which is apparent from reduced phospholipid mobility and morphological changes in fibril bundling. At higher lipid to protein ratios, additional vesicles adsorb along the fibrils. While interactions between lipids and amyloid-protein are generally discussed within the perspective of different protein species adsorbing to and perturbing the lipid membrane, the current work reveals amyloid formation in the presence of lipids as a co-aggregation process. The interaction leads to the formation of lipid-protein co-aggregates with distinct structure, dynamics and morphology compared to assemblies formed by either lipid or protein alone.

Alteration of Plasma Membrane Organization by an Anticancer Lysophosphatidylcholine Analogue Induces Intracellular Acidification and Internalization of Plasma Membrane Transporters in Yeast

The lysophosphatidylcholine analogue edelfosine is a potent antitumor lipid that targets cellular membranes. The underlying mechanisms leading to cell death remain controversial, although two cellular membranes have emerged as primary targets of edelfosine, the plasma membrane (PM) and the endoplasmic reticulum. In an effort to identify conditions that enhance or prevent the cytotoxic effect of edelfosine, we have conducted genome-wide surveys of edelfosine sensitivity and resistance in Saccharomyces cerevisiae presented in this work and the accompanying paper (Cuesta-Marbán, Á., Botet, J., Czyz, O., Cacharro, L. M., Gajate, C., Hornillos, V., Delgado, J., Zhang, H., Amat-Guerri, F., Acuña, A. U., McMaster, C. R., Revuelta, J. L., Zaremberg, V., and Mollinedo, F. (January 23, 2013) J. Biol. Chem. 288,), respectively. Our results point to maintenance of pH homeostasis as a major player in modulating susceptibility to edelfosine with the PM proton pump Pma1p playing a main role. We demonstrate that edelfosine alters PM organization and induces intracellular acidification. Significantly, we show that edelfosine selectively reduces lateral segregation of PM proteins like Pma1p and nutrient H(+)-symporters inducing their ubiquitination and internalization. The biology associated to the mode of action of edelfosine we have unveiled includes selective modification of lipid raft integrity altering pH homeostasis, which in turn regulates cell growth.

Leishmania parasites possess a platelet-activating factor acetylhydrolase important for virulence

Leishmania parasites are intracellular protozoans capable of salvaging and remodeling lipids from the host. To understand the role of lipid metabolism in Leishmania virulence, it is necessary to characterize the enzymes involved in the uptake and turnover of phospholipids. This study focuses on a putative phospholipase A2 (PLA2)/platelet-activating factor acetylhydrolase (PAF-AH) in Leishmania major. In mammals, PAF-AH is a subgroup of PLA2 catalyzing the hydrolysis/inactivation of platelet-activating factor (PAF), a potent mediator of many leukocyte functions. By immunofluorescence microscopy, L. major PLA2/PAF-AH is predominantly localized in the ER. While wild type L. major parasites are able to hydrolyze PAF, this activity is completely absent in the PLA2/PAF-AH-null mutants. Meanwhile, deletion of PLA2/PAF-AH had no significant effect on the turnover of common glycerophospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylglycerol. PLA2/PAF-AH is not required for the growth of L. major parasites in culture, or the production of GPI-anchored virulence factors. Nonetheless, it does play a key role in the mammalian host as the PLA2/PAF-AH null mutants exhibit attenuated virulence in BALB/c mice. In conclusion, these data suggest that Leishmania parasites possess a functional PAF-AH and the degradation of PAF or PAF-like lipids is an important step in infection.

In vitro lipid transfer between lipoproteins and midgut-diverticula in the spider Polybetes pythagoricus

It has been already reported that most hemolymphatic lipids in the spider Polybetes pythagoricus are transported by HDL1 and VHDL lipoproteins. We studied in vitro the lipid transfer among midgut-diverticula (M-diverticula), and either hemolymph or purified lipoproteins as well as between hemolymphatic lipoproteins. M-diverticula and hemolymph were labeled by in vivo (14)C-palmitic acid injection. In vitro incubations were performed between M-diverticula and either hemolymph or isolated lipoproteins. Hemolymph lipid uptake was associated to HDL1 (67%) and VHDL (32%). Release from hemolymph towards M-diverticula showed the opposite trend, VHDL 75% and HDL1 45%. Isolated lipoproteins showed a similar behavior to that observed with whole hemolymph. Lipid transfer between lipoproteins showed that HDL1 transfer more (14)C-lipids to VHDL than vice versa. Only 38% FFA and 18% TAG were transferred from M-diverticula to lipoproteins, while on the contrary 75% and 73% of these lipids, respectively, were taken up from hemolymph. A similar trend was observed regarding lipoprotein phospholipids. This study supports the hypothesis that HDL1 and hemocyanin-containing VHDL are involved in the uptake and release of FFA, phospholipids and triacylglycerols in the spider P. pythagoricus. The data support a directional flow of lipids from HDL1 and VHDL suggesting a mode of lipid transport between lipoproteins and M-diverticula.

A Nonaggregating Surfactant Protein C Mutant Is Misdirected to Early Endosomes and Disrupts Phospholipid Recycling

Interstitial lung disease in both children and adults has been linked to mutations in the lung-specific surfactant protein C (SFTPC) gene. Among these, the missense mutation [isoleucine to threonine at codon 73 = human surfactant protein C (hSP-C(I73T) )] accounts for ∼30% of all described SFTPC mutations. We reported previously that unlike the BRICHOS misfolding SFTPC mutants, expression of hSP-C(I73T) induces lung remodeling and alveolar lipoproteinosis without a substantial Endoplasmic Reticulum (ER) stress response or ER-mediated intrinsic apoptosis. We show here that, in contrast to its wild-type counterpart that is directly routed to lysosomal-like organelles for processing, SP-C(I73T) is misdirected to the plasma membrane and subsequently internalized to the endocytic pathway via early endosomes, leading to the accumulation of abnormally processed proSP-C isoforms. Functionally, cells expressing hSP-C(I73T) demonstrated both impaired uptake and degradation of surfactant phospholipid, thus providing a molecular mechanism for the observed lipid accumulation in patients expressing hSP-C(I73T) through the disruption of normal phospholipid recycling. Our data provide evidence for a novel cellular mechanism for conformational protein-associated diseases and suggest a paradigm for mistargeted proteins involved in the disruption of the endosomal/lysosomal sorting machinery.

StarD7 Gene Expression in Trophoblast Cells: Contribution of SF-1 and Wnt-β-Catenin Signaling

Steroidogenic acute regulatory protein-related lipid transfer domain containing 7 (StarD7) is a poorly characterized member of the steroidogenic acute regulatory protein-related lipid transfer proteins, up-regulated in JEG-3 cells, involved in intracellular transport and metabolism of lipids. Previous studies dealing with the mechanisms underlying the human StarD7 gene expression led us to define the cis-acting regulatory sequences in the StarD7 promoter using as a model JEG-3 cells. These include a functional T cell-specific transcription factor 4 (TCF4) site involved in Wnt-β-catenin signaling. To understand these mechanisms in more depth, we examined the steroidogenic factor 1 (SF-1) contribution to StarD7 expression. Cotransfection experiments in JEG-3 cells point out that the StarD7 promoter is activated by SF-1, and this effect is increased by forskolin. EMSA using JEG-3 nuclear proteins demonstrated that SF-1 binds to the StarD7 promoter. Additionally, chromatin immunoprecipitation analysis indicated that SF-1 and β-catenin are bound in vivo to the StarD7 promoter. Reporter gene assays in combination with mutations in the SF-1 and TCF4 binding sites revealed that the StarD7 promoter is synergistically activated by SF-1 and β-catenin and that the TCF4 binding site (-614/-608) plays an important role in this activation. SF-1 amino acid mutations involved in the physical interaction with β-catenin abolished this activation; thus demonstrating that the contact between the two proteins is necessary for an efficient StarD7 transcriptional induction. Finally, these data suggest that β-catenin could function as a bridge between SF-1 and TCF4 forming a ternary complex, which would stimulate StarD7 expression. The SF-1 and β-catenin pathway convergence on StarD7 expression may have important implications in the phospholipid uptake and transport, contributing to the normal trophoblast development.

Human TMEM30a Promotes Uptake of Antitumor and Bioactive Choline Phospholipids into Mammalian Cells

Antitumor alkylphospholipids initiate apoptosis in transformed HL-60 and Jurkat cells while sparing their progenitors. 1-O-Alkyl-2-carboxymethyl-sn-glycero-3-phosphocholine (Edelfosine) like other short-chained phospholipids--inflammatory platelet-activating factor (PAF) and apoptotic oxidatively truncated phospholipids--are proposed to have intracellular sites of action, yet a conduit for these choline phospholipids into mammalian cells is undefined. Edelfosine is also accumulated by Saccharomyces cerevisiae in a process requiring the membrane protein Lem3p, and the human genome contains a Lem3p homolog TMEM30a. We show that import of choline phospholipids into S. cerevisiae ΔLem3 is partially reconstituted by human TMEM30a and by Lem3p-TMEM30a chimeras, showing the proteins are orthologous. TMEM30a-GFP chimeras expressed in mammalian cells localized in plasma membranes, as well as internal organelles, and ectopic TMEM30a expression promoted uptake of exogenous choline and ethanolamine phospholipids. Short hairpin RNA knockdown of TMEM30a reduced fluorescent choline phospholipid and [(3)H]PAF import. This knockdown also reduced mitochondrial depolarization from exogenous Edelfosine or the mitotoxic oxidatively truncated phospholipid azelaoyl phosphatidylcholine, and the knockdown reduced apoptosis in response to these two phospholipids. These results show that extracellular choline phospholipids with short sn-2 residues can have intracellular roles and sites of metabolism because they are transport substrates for a TMEM30a phospholipid import system. Variation in this mechanism could limit sensitivity to short chain choline phospholipids such as Edelfosine, PAF, and proapoptotic phospholipids.

Synaptic PRG-1 Modulates Excitatory Transmission via Lipid Phosphate-Mediated Signaling

Plasticity related gene-1 (PRG-1) is a brain-specific membrane protein related to lipid phosphate phosphatases, which acts in the hippocampus specifically at the excitatory synapse terminating on glutamatergic neurons. Deletion of prg-1 in mice leads to epileptic seizures and augmentation of EPSCs, but not IPSCs. In utero electroporation of PRG-1 into deficient animals revealed that PRG-1 modulates excitation at the synaptic junction. Mutation of the extracellular domain of PRG-1 crucial for its interaction with lysophosphatidic acid (LPA) abolished the ability to prevent hyperexcitability. As LPA application in vitro induced hyperexcitability in wild-type but not in LPA(2) receptor-deficient animals, and uptake of phospholipids is reduced in PRG-1-deficient neurons, we assessed PRG-1/LPA(2) receptor-deficient animals, and found that the pathophysiology observed in the PRG-1-deficient mice was fully reverted. Thus, we propose PRG-1 as an important player in the modulatory control of hippocampal excitability dependent on presynaptic LPA(2) receptor signaling.