Cerebral Apolipoprotein D Exits the Brain and Accumulates in Peripheral Tissues.

Apolipoprotein D (ApoD) is a secreted lipocalin associated with neuroprotection and lipid metabolism. In rodent, the bulk of its expression occurs in the central nervous system. Despite this, ApoD has profound effects in peripheral tissues, indicating that neural ApoD may reach peripheral organs. We endeavor to determine if cerebral ApoD can reach the circulation and accumulate in peripheral tissues. Three hours was necessary for over 40% of all the radiolabeled human ApoD (hApoD), injected bilaterally, to exit the central nervous system (CNS). Once in circulation, hApoD accumulates mostly in the kidneys/urine, liver, and muscles. Accumulation specificity of hApoD in these tissues was strongly correlated with the expression of lowly glycosylated basigin (BSG, CD147). hApoD was observed to pass through bEnd.3 blood brain barrier endothelial cells monolayers. However, cyclophilin A did not impact hApoD internalization rates in bEnd.3, indicating that ApoD exit from the brain is either independent of BSG or relies on additional cell types. Overall, our data showed that ApoD can quickly and efficiently exit the CNS and reach the liver and kidneys/urine, organs linked to the recycling and excretion of lipids and toxins. This indicated that cerebral overexpression during neurodegenerative episodes may serve to evacuate neurotoxic ApoD ligands from the CNS.

Endogenous retrovirus-encoded Syncytin-2 contributes to exosome-mediated immunosuppression of T cells†.

Modulation of the activation status of immune cell populations during pregnancy depends on placental villous cytotrophoblast (VCT) cells and the syncytiotrophoblast (STB). Failure in the establishment of this immunoregulatory function leads to pregnancy complications. Our laboratory has been studying Syncytin-2 (Syn-2), an endogenous retroviral protein expressed in placenta and on the surface of placental exosomes. This protein plays an important role not only in STB formation through its fusogenic properties, but also through its immunosuppressive domain (ISD). Considering that Syn-2 expression is importantly reduced in preeclamptic placentas, we were interested in addressing its possible immunoregulatory effects on T cells. Activated Jurkat T cells and peripheral blood mononuclear cells (PBMCs) were treated with monomeric or dimerized version of a control or a Syn-2 ISD peptide. Change in phosphorylation levels of ERK1/2 MAP kinases was selectively noted in Jurkat cells treated with the dimerized ISD peptide. Upon incubation with the dimerized Syn-2 ISD peptide, significant reduction in Th1 cytokine production was further demonstrated by ELISA and Human Th1/Th2 Panel Multi-Analyte Flow Assay. To determine if exosome-associated Syn-2 could also be immunosuppressive placental exosomes were incubated with activated Jurkat and PBMCs. Quantification of Th1 cytokines in the supernatants revealed severe reduction in T cell activation. Interestingly, exosomes from Syn-2-silenced VCT incubated with PBMCs were less suppressive when compared with exosome derived from VCT transfected with control small interfering RNA (siRNA). Our results suggest that Syn-2 is an important immune regulator both locally and systemically, via its association with placental exosomes.

HIV-1 Antisense Protein of Different Clades Induces Autophagy and Associates with the Autophagy Factor p62.

The existence of the antisense transcript-encoded HIV-1 antisense protein (ASP) was recently reinforced by in silico analyses providing evidence for recent appearance of this gene in the viral genome. Our previous studies led to the detection of ASP in various cell lines by Western blotting, flow cytometry, and confocal microscopy analyses and reported that it induced autophagy, potentially through multimer formation. Here, our goals were to assess autophagy induction by ASP from different clades and to identify the implicated autophagy factors. We first demonstrated that ASP formed multimers, partly through its amino-terminal region and cysteine residues. Removal of this region was further associated with lower induction of autophagy, as assessed by autophagosome formation. ASPs from different clades (A, B, C, D, and G) were tested next and were detected in monomeric and multimeric forms at various levels, and all induced autophagy (clade A ASP was less efficient), as determined by LC3-II and p62 (SQSTM1) levels. Furthermore, CRISPR-based knockout of ATG5, ATG7, and p62 genes led to increased ASP levels. Confocal microscopy analyses showed that ASP colocalized with p62 and LC3-II in autophagosome-like structures. Coimmunoprecipitation experiments further demonstrated that p62 associated with ASP through its PB1 domain. Interestingly, immunoprecipitation experiments supported the idea that ASP is ubiquitinated and that ubiquitination was modulating its stability. We are thus suggesting that ASP induces autophagy through p62 interaction and that its abundance is controlled by autophagy, in which ubiquitin plays an important role. Understanding the mechanisms underlying ASP degradation is essential to better assess its function.IMPORTANCE In the present study, we provide the first evidence that a new HIV-1 protein termed ASP derived from different clades acts similarly in inducing autophagy, an important cellular process implicated in the degradation of excess or defective cellular material. We have gained further knowledge on the mechanism mediating the activation of autophagy. Our studies have important ramifications in the understanding of viral replication and the pathogenesis associated with HIV-1 in infected individuals. Indeed, autophagy is implicated in antigen presentation during immune response and could thus be rendered inefficient in infected cells, such as dendritic cells. Furthermore, a possible link with HIV-1-associated neurological disorder (HAND) might also be a possible association with the capacity of ASP to induce autophagy. Our studies hence demonstrate the importance in conducting further studies on this protein as it could represent a new interesting target for antiretroviral therapies and vaccine design.

Galectin-1 interacts with the human endogenous retroviral envelope protein syncytin-2 and potentiates trophoblast fusion in humans.

Syncytin (Syn)-2 is an important fusogenic protein that contributes to the formation of the placental syncytiotrophoblast. Galectin (Gal)-1, a soluble lectin, is also involved in trophoblast cell fusion and modulates the interaction of certain retroviral envelopes with their cellular receptor. This study aimed to investigate the association between Syn-2 and Gal-1 during human trophoblast cell fusion. This association was evaluated in vitro on primary villous cytotrophoblasts (vCTBs) and cell lines using recombinant Gal-1 and Syn-2-pseudotyped viruses. Using lactose, a Gal antagonist, and Gal-1-specific small interfering RNA (siRNA) transfections, we confirmed the implication of Gal-1 in vCTBs and BeWo cell fusion, although RT-PCR and ELISA analyses suggested that Gal-1 alone did not induce syncytialization. Infection assays showed a specific and significant effect of Gal-1 on the infectivity of Syn-2-pseudotyped viruses that depended on the expression of major facilitator superfamily domain-containing 2A (MFSD2a). Moreover, Gal-3, another placental Gal, did not modulate the infectivity of Syn-2-positive viruses, strengthening the specific association between Gal-1 and Syn-2. Interestingly, Gal-1 significantly reduced the infectivity of Syn-1-pseudotyped viruses, suggesting the opposite effects of Gal-1 on Syn-1 and -2. Finally, coimmunoprecipitation experiments showed a glycan-dependent interaction between Syn-2-bearing virions and Gal-1. We conclude that Gal-1 specifically interacts with Syn-2 and possibly regulates Syn-2/MFSD2a interaction during syncytialization of trophoblastic cells.-Toudic, C., Vargas, A., Xiao, Y., St-Pierre, G., Bannert, N., Lafond, J., Rassart, É., Sato, S., Barbeau, B. Galectin-1 interacts with the human endogenous retroviral envelope protein syncytin-2 and potentiates trophoblast fusion in humans.

Histone deacetylase 1 interacts with HIV-1 Integrase and modulates viral replication.

BACKGROUND: HIV-1 hijacks the cellular machinery for its own replication through protein-protein interactions between viral and host cell factors. One strategy against HIV-1 infection is thus to target these key protein complexes. As the integration of reverse transcribed viral cDNA into a host cell chromosome is an essential step in the HIV-1 life cycle, catalyzed by the viral integrase and other important host factors, we aimed at identifying new integrase binding partners through a novel approach. METHODS: A LTR-derived biotinylated DNA fragment complexed with the integrase on magnetic beads was incubated with extracts from integrase-expressing 293 T cells. Liquid chromatography-mass spectrometry/mass spectrometry and co-immunoprecipitation/pull-down experiments were used for the identification of binding partners. Transfections of histone deacetylase 1 (HDAC1) expression vectors and/or specific siRNA were conducted in HeLa-CD4 and 293 T cells followed by infection with fully infectious NL4-3 and luciferase-expressing pseudotyped viruses or by proviral DNA transfection. Fully infectious and pseudotyped viruses produced from HDAC1-silenced 293 T cells were tested for their infectivity toward HeLa-CD4 cells, T cell lines and primary CD4+ T cells. Late RT species and integrated viral DNA were quantified by qPCR and infectivity was measured by luciferase activity and p24 ELISA assay. Results were analyzed by the Student's t-test. RESULTS: Using our integrase-LTR bait approach, we successfully identified new potential integrase-binding partners, including HDAC1. We further confirmed that HDAC1 interacted with the HIV-1 integrase in co-immunoprecipitation and pull-down experiments. HDAC1 knockdown in infected HeLa cells was shown to interfere with an early preintegration step of the HIV-1 replication cycle, which possibly involves reverse transcription. We also observed that, while HDAC1 overexpression inhibited HIV-1 expression after integration, HDAC1 knockdown had no effect on this step. In virus producer cells, HDAC1 knockdown had a limited impact on virus infectivity in either cell lines or primary CD4+ T cells. CONCLUSIONS: Our results show that HDAC1 interacts with the HIV-1 integrase and affects virus replication before and after integration. Overall, HDAC1 appears to facilitate HIV-1 replication with a major effect on a preintegration step, which likely occurs at the reverse transcription step.

[Antisense proteins of HTLV viruses]. / Les protéines antisens des virus HTLV.

There are four human T-lymphotropic viruses (HTLV-1, 2, 3, 4) that have emerged from the transmission of simian viruses. HTLV-1 was the first retrovirus to be shown to be responsible for a human pathology. The expression of retroviral genes depends mostly on their 5'LTR, but it was revealed that HTLV have a promoter in their 3'LTR, capable of transcription from the antisense strand of their genome. These transcripts can be translated into proteins named HBZ, APH-2, APH-3 and APH-4. Antisense transcription in HTLV-1 and its encoded protein HBZ have been thoroughly studied and it has been suggested that HBZ plays an important role in viral replication and the development of ATL. Very few studies have been conducted on antisense transcription from the three other viruses, although it is likely that these genes are also implicated in viral replication.

Apolipoprotein D Internalization Is a Basigin-dependent Mechanism.

Apolipoprotein D (apoD), a member of the lipocalin family, is a 29-kDa secreted glycoprotein that binds and transports small lipophilic molecules. Expressed in several tissues, apoD is up-regulated under different stress stimuli and in a variety of pathologies. Numerous studies have revealed that overexpression of apoD led to neuroprotection in various mouse models of acute stress and neurodegeneration. This multifunctional protein is internalized in several cells types, but the specific internalization mechanism remains unknown. In this study, we demonstrate that the internalization of apoD involves a specific cell surface receptor in 293T cells, identified as the transmembrane glycoprotein basigin (BSG, CD147); more particularly, its low glycosylated form. Our results show that internalized apoD colocalizes with BSG into vesicular compartments. Down-regulation of BSG disrupted the internalization of apoD in cells. In contrast, overexpression of basigin in SH-5YSY cells, which poorly express BSG, restored the uptake of apoD. Cyclophilin A, a known ligand of BSG, competitively reduced apoD internalization, confirming that BSG is a key player in the apoD internalization process. In summary, our results demonstrate that basigin is very likely the apoD receptor and provide additional clues on the mechanisms involved in apoD-mediated functions, including neuroprotection.

Characterization and identification of PARM-1 as a new potential oncogene.

BACKGROUND: The Graffi murine retrovirus is a powerful tool to find leukemia associated oncogenes. Using DNA microarrays, we recently identified several genes specifically deregulated in T- and B-leukemias induced by this virus. RESULTS: In the present study, probsets associated with T-CD8+ leukemias were analyzed and we validated the expression profile of the Parm-1 gene. PARM-1 is a member of the mucin family. We showed that human PARM-1 is an intact secreted protein accumulating predominantly, such as murine PARM-1, at the Golgi and in the early and late endosomes. PARM-1 colocalization with α-tubulin suggests that its trafficking within the cell involves the microtubule cytoskeleton. Also, the protein co-localizes with caveolin-1 which probably mediates its internalization. Transient transfection of both mouse and human Parm-1 cDNAs conferred anchorage- and serum-independent growth and enhanced cell proliferation. Moreover, deletion mutants of human PARM-1 without either extracellular or cytoplasmic portions seem to retain the ability to induce anchorage-independent growth of NIH/3T3 cells. In addition, PARM-1 increases ERK1/2, but more importantly AKT and STAT3 phosphorylation. CONCLUSIONS: Our results strongly suggest the oncogenic potential of PARM-1.

Decreasing stearoyl-CoA desaturase-1 expression inhibits ß-catenin signaling in breast cancer cells.

Stearoyl-CoA desaturase-1 (SCD1) is an endoplasmic reticulum anchored enzyme catalyzing the synthesis of monounsaturated fatty acids, mainly palmytoleyl-CoA and oleyl-CoA. Recent studies have revealed a function for SCD1 in the modulation of signaling processes related to cell proliferation, survival and transformation to cancer. We used MCF7 and MDA-MB-231 cells to analyze the role of SCD1 in the metastatic acquisition of breast cancer cells. Silencing SCD1 expression in breast cancer cells has no effect on cell viability but the levels of cell proliferation, cell cycle genes' expressions and the phosphorylation state of ERK1/2 MAPK are significantly reduced. Decreasing SCD1 expression also reduces the level of GSK3 phosphorylation, indicating higher activity of the kinase. Using cells fractionation, immunofluorescence and a ß-catenin/TCF-responsive reporter construct, we demonstrate that lowering SCD1 expression leads to a decrease of ß-catenin amounts within the nucleus and to inhibition of its transactivation capacity. Moreover, MDA-MB-231 cells transfected with the SCD1 siRNA show a lower invasive potential than the control cells. Taken together, our data demonstrate that low SCD1 expression is associated with a decrease in the proliferation rate of breast cancer cells associated with a decrease in ERK1/2 activation. SCD1 silencing also inhibits GSK3 phosphorylation, lowering ß-catenin translocation to the nucleus, and, subsequently, its transactivation capacity and the expression of its target genes. Finally, we show that silencing SCD1 impairs the epithelial to mesenchymal transition-like behavior of the cells, a characteristic of metastatic breast cancer.

Gene profiling of Graffi murine leukemia virus-induced lymphoid leukemias: identification of leukemia markers and Fmn2 as a potential oncogene.

The Graffi murine leukemia virus induces a large spectrum of leukemias in mice and thus provides a good model to compare the transcriptome of all types of leukemias. We analyzed the gene expression profiles of both T and B leukemias induced by the virus with DNA microarrays. Given that we considered that a 4-fold change in expression level was significant, 388 probe sets were associated to B, to T, or common to both leukemias. Several of them were not yet associated with lymphoid leukemia. We confirmed specific deregulation of Fmn2, Arntl2, Bfsp2, Gfra2, Gpm6a, and Gpm6b in B leukemia, of Nln, Fbln1, and Bmp7 in T leukemias, and of Etv5 in both leukemias. More importantly, we show that the mouse Fmn2 induced an anchorage-independent growth, a drastic modification in cell shape with a concomitant disruption of the actin cytoskeleton. Interestingly, we found that human FMN2 is overexpressed in approximately 95% of pre-B acute lymphoblastic leukemia with the highest expression levels in patients with a TEL/AML1 rearrangement. These results, surely related to the role of FMN2 in meiotic spindle maintenance, suggest its important role in leukemogenesis. Finally, we propose a new panel of genes potentially involved in T and/or B leukemias.

Apolipoprotein D in Oxidative Stress and Inflammation.

Apolipoprotein D (ApoD) is lipocalin able to bind hydrophobic ligands. The APOD gene is upregulated in a number of pathologies, including Alzheimer's disease, Parkinson's disease, cancer, and hypothyroidism. Upregulation of ApoD is linked to decreased oxidative stress and inflammation in several models, including humans, mice, Drosophila melanogaster and plants. Studies suggest that the mechanism through which ApoD modulates oxidative stress and regulate inflammation is via its capacity to bind arachidonic acid (ARA). This polyunsaturated omega-6 fatty acid can be metabolised to generate large variety of pro-inflammatory mediators. ApoD serves as a sequester, blocking and/or altering arachidonic metabolism. In recent studies of diet-induced obesity, ApoD has been shown to modulate lipid mediators derived from ARA, but also from eicosapentaenoic acid and docosahexaenoic acid in an anti-inflammatory way. High levels of ApoD have also been linked to better metabolic health and inflammatory state in the round ligament of morbidly obese women. Since ApoD expression is upregulated in numerous diseases, it might serve as a therapeutic agent against pathologies aggravated by OS and inflammation such as many obesity comorbidities. This review will present the most recent findings underlying the central role of ApoD in the modulation of both OS and inflammation.

Galectin-1 Modulates the Fusogenic Activity of Placental Endogenous Retroviral Envelopes.

Syncytin-1 and -2 are glycoproteins encoded by human endogenous retrovirus (hERV) that, through their fusogenic properties, are needed for the formation of the placental syncytiotrophoblast. Previous studies suggested that these proteins, in addition to the EnvP(b) envelope protein, are also involved in other cell fusion events. Since galectin-1 is a ß-galactoside-binding protein associated with cytotrophoblast fusion during placental development, we previously tested its effect on Syncytin-mediated cell fusion and showed that this protein differently modulates the fusogenic potential of Syncytin-1 and -2. Herein, we were interested in comparing the impact of galectin-1 on hERV envelope proteins in different cellular contexts. Using a syncytium assay, we first demonstrated that galectin-1 increased the fusion of Syncytin-2- and EnvP(b)-expressing cells. We then tested the infectivity of Syncytin-1 and -2 vs. VSV-G-pseudotyped viruses toward Cos-7 and various human cell lines. In the presence of galectin-1, infection of Syncytin-2-pseudotyped viruses augmented for all cell lines. In contrast, the impact of galectin-1 on the infectivity of Syncytin-1-pseudotyped viruses varied, being cell- and dose-dependent. In this study, we report the functional associations between three hERV envelope proteins and galectin-1, which should provide information on the fusogenic activity of these proteins in the placenta and other biological and pathological processes.

A newly identified interaction between nucleolar NPM1/B23 and the HTLV-I basic leucine zipper factor in HTLV-1 infected cells.

Human T-cell leukemia virus type 1 is the causative agent of HTLV-1-associated myelopathy/tropical spastic paraparesis and adult T-cell leukemia-lymphoma (ATL). The HTLV-1 basic leucine zipper factor (HBZ) has been associated to the cancer-inducing properties of this virus, although the exact mechanism is unknown. In this study, we identified nucleophosmin (NPM1/B23) as a new interaction partner of HBZ. We show that sHBZ and the less abundant uHBZ isoform interact with nucleolar NPM1/B23 in infected cells and HTLV-1 positive patient cells, unlike equivalent antisense proteins of related non-leukemogenic HTLV-2, -3 and-4 viruses. We further demonstrate that sHBZ association to NPM1/B23 is sensitive to RNase. Interestingly, sHBZ was shown to interact with its own RNA. Through siRNA and overexpression experiments, we further provide evidence that NPM1/B23 acts negatively on viral gene expression with potential impact on cell transformation. Our results hence provide a new insight over HBZ-binding partners in relation to cellular localization and potential function on cell proliferation and should lead to a better understanding of the link between HBZ and ATL development.

Characterization of nuclear factors modulating the apolipoprotein D promoter during growth arrest: implication of PARP-1, APEX-1 and ERK1/2 catalytic activities.

Human Apolipoprotein D (apoD) is upregulated under several stress conditions and pathological situations such as neurodegenerative diseases and cancers. We previously showed that apoD mRNA expression is induced in growth-arrested cells and demonstrated the specific binding of nuclear proteins to the region -514 to -475 of the promoter. Such region contains a pair of Serum Responsive Elements (SRE), an Ets-Binding Site (EBS) and a Glucocorticoid Responsive Element (GRE). In this study, we show that Parp-1, HnRNP-U, CBF-A, BUB-3, Kif4, APEX-1 and Ifi204 bind these regulatory elements of the apoD promoter. Specific binding of HnRNP-U and Parp-1 was confirmed by Electrophoretic Mobility Shift Assay (EMSA). In a biotin pull-down assay, Kif4 and BUB-3 bind preferentially the SRE1 and the EBS-GRE sites, respectively, while APEX-1 seems recruited indirectly to these elements. We found that the mRNA expression of some of these binding factors is upregulated in growth-arrested cells and that these proteins also transactivate the apoD promoter. In agreement with these results, mutants of APEX-1 and of Parp-1 defective for their DNA-binding and catalytic activities could not transactivate the promoter. The knockdown of Parp-1 and HnRNP-U and the use of specific inhibitors of MEK1/2 and of Parp-1 also inhibited the induction of apoD gene expression. Moreover, ERK1/2 was found activated in a biphasic manner post serum-starvation and the inhibition of Parp-1 causes a sustained activation of ERK2 but not ERK1 for up to 2h. Altogether, these findings demonstrate the importance of Parp-1, APEX-1 and ERK1/2 catalytic activities in the growth arrest-induced apoD gene expression.

Apolipoprotein D alters the early transcriptional response to oxidative stress in the adult cerebellum.

The lipocalin Apolipoprotein D (ApoD), known to protect the nervous system against oxidative stress (OS) in model organisms, is up-regulated early in the mouse brain in response to the ROS generator paraquat. However, the processes triggered by this up-regulation have not been explored. We present here a study of the effect of ApoD on the early transcriptional changes upon OS in the mouse cerebellum using microarray profiling. ApoD-KO and transgenic mice over-expressing ApoD in neurons are compared to wild-type controls. In control conditions, ApoD affects the transcriptional profile of neuron and oligodendrocyte-specific genes involved in neuronal excitability, synaptic function, and myelin homeostasis. When challenged with paraquat, the absence of ApoD modifies the response of genes mainly related to OS management and myelination. Interestingly, the over-expression of ApoD in neurons almost completely abolishes the early transcriptional response to OS. We independently evaluate the expression of protein kinase Cδ, a gene up-regulated by OS only in the ApoD-KO cerebellum, and find it over-expressed in cultured ApoD-KO primary astrocytes, which points to a role for ApoD in astrocyte-microglia signaling. Our results support the hypothesis that ApoD is necessary for a proper response of the nervous system against physiological and pathological OS.

Importin alpha3 interacts with HIV-1 integrase and contributes to HIV-1 nuclear import and replication.

HIV-1 employs the cellular nuclear import machinery to actively transport its preintegration complex (PIC) into the nucleus for integration of the viral DNA. Several viral karyophilic proteins and cellular import factors have been suggested to contribute to HIV-1 PIC nuclear import and replication. However, how HIV interacts with different cellular machineries to ensure efficient nuclear import of its preintegration complex in dividing and nondividing cells is still not fully understood. In this study, we have investigated different importin alpha (Impalpha) family members for their impacts on HIV-1 replication, and we demonstrate that short hairpin RNA (shRNA)-mediated Impalpha3 knockdown (KD) significantly impaired HIV infection in HeLa cells, CD4(+) C8166 T cells, and primary macrophages. Moreover, quantitative real-time PCR analysis revealed that Impalpha3-KD resulted in significantly reduced levels of viral 2-long-terminal repeat (2-LTR) circles but had no effect on HIV reverse transcription. All of these data indicate an important role for Impalpha3 in HIV nuclear import. In an attempt to understand how Impalpha3 participates in HIV nuclear import and replication, we first demonstrated that the HIV-1 karyophilic protein integrase (IN) was able to interact with Impalpha3 both in a 293T cell expression system and in HIV-infected CD4(+) C8166 T cells. Deletion analysis suggested that a region (amino acids [aa] 250 to 270) in the C-terminal domain of IN is involved in this viral-cellular protein interaction. Overall, this study demonstrates for the first time that Impalpha3 is an HIV integrase-interacting cofactor that is required for efficient HIV-1 nuclear import and replication in both dividing and nondividing cells.

ApoD, a glia-derived apolipoprotein, is required for peripheral nerve functional integrity and a timely response to injury.

Glial cells are a key element to the process of axonal regeneration, either promoting or inhibiting axonal growth. The study of glial derived factors induced by injury is important to understand the processes that allow or preclude regeneration, and can explain why the PNS has a remarkable ability to regenerate, while the CNS does not. In this work we focus on Apolipoprotein D (ApoD), a Lipocalin expressed by glial cells in the PNS and CNS. ApoD expression is strongly induced upon PNS injury, but its role has not been elucidated. Here we show that ApoD is required for: (1) the maintenance of peripheral nerve function and tissue homeostasis with age, and (2) an adequate and timely response to injury. We study crushed sciatic nerves at two ages using ApoD knock-out and transgenic mice over-expressing human ApoD. The lack of ApoD decreases motor nerve conduction velocity and the thickness of myelin sheath in intact nerves. Following injury, we analyze the functional recovery, the cellular processes, and the protein and mRNA expression profiles of a group of injury-induced genes. ApoD helps to recover locomotor function after injury, promoting myelin clearance, and regulating the extent of angiogenesis and the number of macrophages recruited to the injury site. Axon regeneration and remyelination are delayed without ApoD and stimulated by excess ApoD. The mRNA and protein expression profiles reveal that ApoD is functionally connected in an age-dependent manner to specific molecular programs triggered by injury.

Evidence for transcriptional regulation of the glucose-6-phosphate transporter by HIF-1alpha: Targeting G6PT with mumbaistatin analogs in hypoxic mesenchymal stromal cells.

Mesenchymal stromal cell (MSC) markers are expressed on brain tumor-initiating cells involved in the development of hypoxic glioblastoma. Given that MSCs can survive hypoxia and that the glucose-6-phosphate transporter (G6PT) provides metabolic control that contributes to MSC mobilization and survival, we investigated the effects of low oxygen (1.2% O(2)) exposure on G6PT gene expression. We found that MSCs significantly expressed G6PT and the glucose-6-phosphatase catalytic subunit beta, whereas expression of the glucose-6-phosphatase catalytic subunit alpha and the islet-specific glucose-6-phosphatase catalytic subunit-related protein was low to undetectable. Analysis of the G6PT promoter sequence revealed potential binding sites for hypoxia inducible factor (HIF)-1alpha and for the aryl hydrocarbon receptor (AhR) and its dimerization partner, the AhR nuclear translocator (ARNT), AhR:ARNT. In agreement with this, hypoxia and the hypoxia mimetic cobalt chloride induced the expression of G6PT, vascular endothelial growth factor (VEGF), and HIF-1alpha. Gene silencing of HIF-1alpha prevented G6PT and VEGF induction in hypoxic MSCs whereas generation of cells stably expressing HIF-1alpha resulted in increased endogenous G6PT gene expression. A semisynthetic analog of the polyketide mumbaistatin, a potent G6PT inhibitor, specifically reduced MSC-HIF-1alpha cell survival. Collectively, our data suggest that G6PT may account for the metabolic flexibility that enables MSCs to survive under conditions characterized by hypoxia and could be specifically targeted within developing tumors.

SCD1 activity promotes cell migration via a PLD-mTOR pathway in the MDA-MB-231 triple-negative breast cancer cell line.

BACKGROUND: Breast cancer is the most common cancer in women. Despite high survival rates in Western countries, treatments are less effective in metastatic cases and triple-negative breast cancer (TNBC) patient survival is the shortest across breast cancer subtypes. High expression levels of stearoyl-CoA desaturase-1 (SCD1) have been reported in breast cancer. The SCD1 enzyme catalyzes the formation of oleic acid (OA), a lipid stimulating the migration of metastatic breast cancer cells. Phospholipase activity is also implicated in breast cancer metastasis, notably phospholipase D (PLD). METHODS: Kaplan-Meier survival plots generated from gene expression databases were used to analyze the involvement of SCD1 and PLD in several cancer subtypes. SCD1 enzymatic activity was modulated with a pharmaceutical inhibitor or by OA treatment (to mimic SCD1 over-activity) in three breast cancer cell lines: TNBC-derived MDA-MB-231 cells as well as non-TNBC MCF-7 and T47D cells. Cell morphology and migration properties were characterized by various complementary methods. RESULTS: Our survival analyses suggest that SCD1 and PLD2 expression in the primary tumor are both associated to metastasis-related morbid outcomes in breast cancer patients. We show that modulation of SCD1 activity is associated with the modification of TNBC cell migration properties, including changes in speed, direction and cell morphology. Cell migration properties are regulated by SCD1 activity through a PLD-mTOR/p70S6K signaling pathway. These effects are not observed in non-TNBC cell lines. CONCLUSION: Our results establish a key role for the lipid desaturase SCD1 and delineate an OA-PLD-mTOR/p70S6K signaling pathway in TNBC-derived MDA-MB-231 cell migration.

Apolipoprotein D.

ApoD is a 25 to 30 kDa glycosylated protein, member of the lipocalin superfamily. As a transporter of several small hydrophobic molecules, its known biological functions are mostly associated to lipid metabolism and neuroprotection. ApoD is a multi-ligand, multi-function protein that is involved lipid trafficking, food intake, inflammation, antioxidative response and development and in different types of cancers. An important aspect of ApoD's role in lipid metabolism appears to involve the transport of arachidonic acid, and the modulation of eicosanoid production and delivery in metabolic tissues. ApoD expression in metabolic tissues has been associated positively and negatively with insulin sensitivity and glucose homeostasis in a tissue dependent manner. ApoD levels rise considerably in association with aging and neuropathologies such as Alzheimer's disease, stroke, meningoencephalitis, moto-neuron disease, multiple sclerosis, schizophrenia and Parkinson's disease. ApoD is also modulated in several animal models of nervous system injury/pathology.