Françoise Hullin-Matsuda

Novel lipogenic enzyme ELOVL7 is involved in prostate cancer growth through saturated long-chain fatty acid metabolism.

A number of epidemiologic studies have indicated a strong association between dietary fat intake and prostate cancer development, suggesting that lipid metabolism plays some important roles in prostate carcinogenesis and its progression. In this study, through our genome-wide gene expression analysis of clinical prostate cancer cells, we identified a novel lipogenic gene, ELOVL7, coding a possible long-chain fatty acid elongase, as overexpressed in prostate cancer cells. ELOVL7 expression is regulated by the androgen pathway through SREBP1, as well as other lipogenic enzymes. Knockdown of ELOVL7 resulted in drastic attenuation of prostate cancer cell growth, and it is notable that high-fat diet promoted the growth of in vivo tumors of ELOVL7-expressed prostate cancer. In vitro fatty acid elongation assay and fatty acid composition analysis indicated that ELOVL7 was preferentially involved in fatty acid elongation of saturated very-long-chain fatty acids (SVLFA, C20:0 approximately ). Lipid profiles showed that knockdown of ELOVL7 in prostate cancer cells affected SVLFAs in the phospholipids and the neutral lipids, such as cholesterol ester. Focusing on cholesterol ester as a source of de novo steroid synthesis, we show that ELOVL7 affected de novo androgen synthesis in prostate cancer cells. These findings suggest that EVOLV7 could be involved in prostate cancer growth and survival through the metabolism of SVLFAs and their derivatives, could be a key molecule to elucidate the association between fat dietary intake and prostate carcinogenesis, and could also be a promising molecular target for development of new therapeutic or preventive strategies for prostate cancers.

A Role for Sphingomyelin-Rich Lipid Domains in the Accumulation of Phosphatidylinositol-4,5-Bisphosphate to the Cleavage Furrow during Cytokinesis

ABSTRACT Cytokinesis is a crucial step in the creation of two daughter cells by the formation and ingression of the cleavage furrow. Here, we show that sphingomyelin (SM), one of the major sphingolipids in mammalian cells, is required for the localization of phosphatidylinositol-4,5-bisphosphate (PIP2) to the cleavage furrow during cytokinesis. Real-time observation with a labeled SM-specific protein, lysenin, revealed that SM is concentrated in the outer leaflet of the furrow at the time of cytokinesis. Superresolution fluorescence microscopy analysis indicates a transbilayer colocalization between the SM-rich domains in the outer leaflet and PIP2-rich domains in the inner leaflet of the plasma membrane. The depletion of SM disperses PIP2 and inhibits the recruitment of the small GTPase RhoA to the cleavage furrow, leading to abnormal cytokinesis. These results suggest that the formation of SM-rich domains is required for the accumulation of PIP2 to the cleavage furrow, which is a prerequisite for the proper translocation of RhoA and the progression of cytokinesis.

De novo biosynthesis of the late endosome lipid, bis(monoacylglycero )phosphate

Bis(monoacylglycero)phosphate (BMP) is a unique lipid enriched in the late endosomes participating in the trafficking of lipids and proteins through this organelle. The de novo biosynthesis of BMP has not been clearly demonstrated. We investigated whether phosphatidylglycerol (PG) and cardiolipin (CL) could serve as precursors of de novo BMP synthesis using two different cellular models: CHO cells deficient in phosphatidylglycerophosphate (PGP) synthase, the enzyme responsible for the first step of PG synthesis; and human lymphoblasts from patients with Barth syndrome (BTHS), characterized by mutations in tafazzin, an enzyme implicated in the deacylation-reacylation cycle of CL. The biosynthesis of both PG and BMP was reduced significantly in the PGP synthase-deficient CHO mutants. Furthermore, overexpression of PGP synthase in the deficient mutants induced an increase of BMP biosynthesis. In contrast to CHO mutants, BMP biosynthesis and its fatty acid composition were not altered in BTHS lymphoblasts. Our results thus suggest that in mammalian cells, PG, but not CL, is a precursor of the de novo biosynthesis of BMP. Despite the decrease of de novo synthesis, the cellular content of BMP remained unchanged in CHO mutants, suggesting that other pathway(s) than de novo biosynthesis are also used for BMP synthesis.

Visualization of the heterogeneous membrane distribution of sphingomyelin associated with cytokinesis, cell polarity, and sphingolipidosis

Sphingomyelin (SM) is a major sphingolipid in mammalian cells and is reported to form specific lipid domains together with cholesterol. However, methods to examine the membrane distribution of SM are limited. We demonstrated in model membranes that fluorescent protein conjugates of 2 specific SM‐binding toxins, lysenin (Lys) and equinatoxin II (EqtII), recognize different membrane distributions of SM; Lys exclusively binds clustered SM, whereas EqtII preferentially binds dispersed SM. Freeze‐fracture immunoelectron microscopy showed that clustered but not dispersed SM formed lipid domains on the cell surface. Glycolipids and the membrane concentration of SM affect the SM distribution pattern on the plasma membrane. Using derivatives of Lys and EqtII as SM distribution‐sensitive probes, we revealed the exclusive accumulation of SM clusters in the midbody at the time of cytokinesis. Interestingly, apical membranes of differentiated epithelial cells exhibited dispersed SM distribution, whereas SM was clustered in basolateral membranes. Clustered but not dispersed SM was absent from the cell surface of acid sphingomyelinase‐deficient Niemann‐Pick type A cells. These data suggest that both the SM content and membrane distribution are crucial for pathophysiological events bringing therapeutic perspective in the role of SM membrane distribution.—Makino, A., Abe, M., Murate, M., Inaba, T., Yilmaz, N., Hullin‐Matsuda, F., Kishimoto, T., Schieber, N. L., Taguchi, T., Arai, H., Anderluh, G., Parton, R. G., Kobayashi, T. Visualization of the heterogeneous membrane distribution of sphingomyelin associated with cytokinesis, cell polarity, and sphingolipidosis. FASEB J. 29, 477‐493 (2015). www.fasebj.org

Monitoring the distribution and dynamics of signaling microdomains in living cells with lipid-specific probes.

Abstract.Specialized lipid microdomains in the cell plasma membrane, referred to as ‘lipid rafts’ are enriched in sphingolipids and cholesterol and have drawn considerable interest as platforms for the recruitment of signaling molecules. Despite numerous biochemical and cellular studies, debate persists on the size, lifetime and even the existence of lipid rafts, emphasizing the need for reliable lipid probes to study in situ membrane lipid organization. In this review, we summarize our recent data on living cells using two specific probes of raft components: lysenin, a sphingomyelin- binding protein and the fluorescein ester of poly(ethyleneglycol)cholesteryl ether that labels cholesterol-rich domains. Sphingomyelin-rich domains that are spatially and functionally distinct from the GM1 ganglioside-rich domains were found at the plasma membrane of Jurkat T cells. In addition, the dynamics of cholesterol-rich domains could be monitored at the cell surface as well as inside the cells.

Lipid compartmentalization in the endosome system.

Lipids play an essential role in the structure of the endosomal membranes as well as in their dynamic rearrangement during the transport of internalized cargoes along the endocytic pathway. In this review, we discuss the function of endosomal lipids mainly in mammalian cells, focusing on two well-known components of the lipid rafts, sphingomyelin and cholesterol, as well as on three anionic phospholipids, phosphatidylserine, polyphosphoinositides and the atypical phospholipid, bis(monoacylglycero)phosphate/lysobisphosphatidic acid. We detail the structure, metabolism, distribution and role of these lipids in the endosome system as well as their importance in pathological conditions where modification of the endosomal membrane flow can lead to various diseases such as lipid-storage diseases, myopathies and neuropathies.

Limonoid Compounds Inhibit Sphingomyelin Biosynthesis by Preventing CERT Protein-dependent Extraction of Ceramides from the Endoplasmic Reticulum

Background: Pharmacological inhibitors of sphingolipid metabolism and transport are useful for both biological and therapeutic research. Results: High throughput microscopy-based screening identified limonoids as inhibitors of sphingomyelin biosynthesis by preventing the membrane extraction of ceramide. Conclusion: Some therapeutic properties of limonoids might be related to their effects on ceramide metabolism. Significance: This study provides insights into the role of the ceramide domains in sphingolipid metabolism. To identify novel inhibitors of sphingomyelin (SM) metabolism, a new and selective high throughput microscopy-based screening based on the toxicity of the SM-specific toxin, lysenin, was developed. Out of a library of 2011 natural compounds, the limonoid, 3-chloro-8β-hydroxycarapin-3,8-hemiacetal (CHC), rendered cells resistant to lysenin by decreasing cell surface SM. CHC treatment selectively inhibited the de novo biosynthesis of SM without affecting glycolipid and glycerophospholipid biosynthesis. Pretreatment with brefeldin A abolished the limonoid-induced inhibition of SM synthesis suggesting that the transport of ceramide (Cer) from the endoplasmic reticulum to the Golgi apparatus is affected. Unlike the Cer transporter (CERT) inhibitor HPA-12, CHC did not change the transport of a fluorescent short chain Cer analog to the Golgi apparatus or the formation of fluorescent and short chain SM from the corresponding Cer. Nevertheless, CHC inhibited the conversion of de novo synthesized Cer to SM. We show that CHC specifically inhibited the CERT-mediated extraction of Cer from the endoplasmic reticulum membranes in vitro. Subsequent biochemical screening of 21 limonoids revealed that some of them, such as 8β-hydroxycarapin-3,8-hemiacetal and gedunin, which exhibits anti-cancer activity, inhibited SM biosynthesis and CERT-mediated extraction of Cer from membranes. Model membrane studies suggest that 8β-hydroxycarapin-3,8-hemiacetal reduced the miscibility of Cer with membrane lipids and thus induced the formation of Cer-rich membrane domains. Our study shows that certain limonoids are novel inhibitors of SM biosynthesis and suggests that some biological activities of these limonoids are related to their effect on the ceramide metabolism.

Sphingomyelin regulates the transbilayer movement of diacylglycerol in the plasma membrane of Madin-Darby canine kidney cells

Diacylglycerol (DAG) is a key component in lipid metabolism and signaling. Previous model membrane studies using DAG analogs suggest their rapid membrane transbilayer movement. However, little is known about the DAG distribution and dynamics in cell membranes. Using live‐cell fluorescence microscopy, we monitored the transbilayer movement of DAG with the yellow fluorescent protein‐tagged C1AB domain from protein kinase C‐γ (EYFP‐C1AB), which selectively binds DAG. When HeLa cells were treated with Bacillus cereus phospholipase C (Bc‐PLC) to produce DAG on the outer leaflet of the plasma membrane, intracellularly expressed EYFP‐C1AB probe accumulated at the plasma membrane, indicating the transbilayer movement of the outer leaflet DAG to the inner leaflet. This Bc‐PLC‐induced translocation of EYFP‐C1AB probe to the plasma membrane was not observed in the sphingolipid‐enriched plasma membrane of Madin‐Darby canine kidney cells, but was recovered after cell treatment with sphingomyelinase or preincubation with an inhibitor of sphingolipid biosynthesis. The inhibitory effect of sphingomyelin (SM) on the transbilayer movement of DAG was reproduced in model membranes using a fluorescent short‐chain DAG analog. These results demonstrate that the SM content on the outer leaflet regulates the transbilayer movement of DAG in the plasma membrane, thus providing new insights into the dynamics of DAG in cell pathophysiology.—Ueda, Y., Makino, A., Murase‐Tamada, K., Sakai, S., Inaba, T., Hullin‐Matsuda, F., Kobayashi, T., Sphingomyelin regulates the transbilayer movement of diacylglycerol in the plasma membrane of Madin‐Darby canine kidney cells. FASEB J. 27, 3284–3297 (2013). www.fasebj.org

Selective incorporation of docosahexaenoic acid into lysobisphosphatidic acid in cultured THP-1 macrophages.

Lysobisphosphatidic acid (LBPA) is highly accumulated in specific domains of the late endosome and is involve in the biogenesis and function of this organelle. Little is known about the biosynthesis and metabolism of this lipid. We examined its FA composition and the incorporation of exogenous FA into LBPA in the human monocytic leukemia cell line THP-1. The LBPA FA composition in THP-1 cells exhibits an elevated amount of oleic acid (18∶1n−9) and enerichment of PUFA, especially DHA (22∶6n−3). DHA supplemented to the medium was efficiently incorporated into LBPA. In contrast, arachidonic acid (20∶4n−6) was hardly esterified to LBPA under the same experimental conditions. The turnover of DHA in LBPA was similar to that in other phospholipids. Specific incorporation of DHA into LBPA was also observed in baby hamster kidney fibroblasts, although LBPA in these cells contains very low endogenous levels of DHA in normal growth conditions. Our results, together with published observations, suggest that the specific incorporation of DHA into LBPA is a common phenomenon in mammalian cells. The physiological significance of DHA-enriched LBPA is discussed.

Enterophilins, a New Family of Leucine Zipper Proteins Bearing a B30.2 Domain and Associated with Enterocyte Differentiation

Enterocyte terminal differentiation occurs at the crypt-villus junction through the transcriptional activation of cell-specific genes, many of which code for proteins of the brush border membrane such as intestinal alkaline phosphatase, sucrase-isomaltase, or the microvillar structural protein villin. Several studies have shown that this sharp increase in specific mRNA levels is intimately associated with arrest of cell proliferation. We isolated several clones from a guinea pig intestine cDNA library. They encode new proteins characterized by an original structure associating a carboxyl-terminal B30.2/RFP-like domain and a long leucine zipper at the amino terminus. The first member of this novel gene family codes for a 65-kDa protein termed enterophilin-1, which is specifically expressed in enterocytes before their final differentiation. Enterophilin-1 is the most abundant in the small intestine but is still present in significant amounts in colonic enterocytes. In Caco-2 cells, a similar 65-kDa protein was recognized by a specific anti-enterophilin-1 antibody, and its expression was positively correlated with cell differentiation status. In addition, transfection of HT-29 cells with enterophilin-1 full-length cDNA slightly inhibited cell growth and promoted an increase in alkaline phosphatase activity. Taken together, these data identify enterophilins as a new family of proteins associated with enterocyte differentiation.