apoD Gene Expression Research Articles

Control of the neuroprotective Lipocalin Apolipoprotein D expression by alternative promoter regions and differentially expressed mRNA 5’ UTR variants

The Lipocalin Apolipoprotein D (ApoD) is one of the few genes consistently overexpressed in the aging brain, and in most neurodegenerative and psychiatric diseases. Its functions include metabolism regulation, myelin management, neuroprotection, and longevity regulation. Knowledge of endogenous regulatory mechanisms controlling brain disease-triggered ApoD expression is relevant if we want to boost pharmacologically its neuroprotecting potential. In addition to classical transcriptional control, Lipocalins have a remarkable variability in mRNA 5'UTR-dependent translation efficiency. Using bioinformatic analyses, we uncover strong selective pressures preserving ApoD 5'UTR properties, indicating unexpected functional conservation. PCR amplifications demonstrate the production of five 5'UTR variants (A-E) in mouse ApoD, with diverse expression levels across tissues and developmental stages. Importantly, Variant E is specifically expressed in the oxidative stress-challenged brain. Predictive analyses of 5'UTR secondary structures and enrichment in elements restraining translation, point to Variant E as a tight regulator of ApoD expression. We find two genomic regions conserved in human and mouse ApoD: a canonical (α) promoter region and a previously unknown region upstream of Variant E that could function as an alternative mouse promoter (β). Luciferase assays demonstrate that both α and β promoter regions can drive expression in cultured mouse astrocytes, and that Promoter β activity responds proportionally to incremental doses of the oxidative stress generator Paraquat. We postulate that Promoter β works in association with Variant E 5'UTR as a regulatory tandem that organizes ApoD gene expression in the nervous system in response to oxidative stress, the most common factor in aging and neurodegeneration.

Hippocampal Up-Regulation of Apolipoprotein D in a Rat Model of Maternal Hypo- and Hyperthyroidism: Implication of Oxidative Stress.

Thyroid disorders impair various functions of the hippocampus where thyroid hormone receptors are localized in the brain. Hyper and hypothyroidism are associated with large changes in brain oxidative stress. Apolipoprotein D (APOD) is a conserved glycoprotein that increased in response to oxidative stress in the brain and has been suggested function as an antioxidant in the brain. Thus, the goal of this work was to explore the effect of maternal hypo- and hyperthyroidism on the Apod expression in the pup's brain regarding changes in oxidative stress. For induction hypo and hyperthyroidism in adult female rats, 100ppm propylthiouracil (PTU) and 8ppm levothyroxine administrated 1month before copulation to the week 3 after delivery in drinking water. The hippocampal region of rat pups was isolated and used for immunohistochemistry and quantitative RT-PCR on postnatal day (PND)5, PND10 and PND20. Results revealed that APOD over-expressed in both hypo- and hyperthyroid groups on PND5, PND10, and PND20. There was a proportional increase between the Apod expression and oxidative stress in the hyperthyroid group but not the hypothyroid in different days. Regarding the wide functions of thyroid hormones, oxidative stress does not suggest to be the only mechanism that involves Apod gene expression in thyroid disturbances.

Molecular Identification and Expression Patterns of Apolipoprotein D FromBombyx mori(Lepidoptera: Bombycidae) in Response to Oxidative Stress and Bacterial Challenge

Apolipoprotein D (ApoD) is an essential protein for insect internal circulation because of its role in lipid transportation. In this study, we cloned and characterized ApoD and analyzed its expression characteristics in Bombyx mori (L.) (Lepidoptera: Bombycidae). The ApoD cDNA fragment, which has an open reading frame of 522 bp with a lipocalin domain (PF08212), encodes 174 amino acids. The deduced ApoD protein sequence has 28.2% identity to its ortholog in Mus musculus and 27.8% identity to its ortholog GLaz in Drosophila melanogaster . Several putative cis-acting elements related to development were also examined. Using quantitative real time polymerase chain reaction (RT-PCR), the ApoD gene expression was detected in the head and silk glands. Western blotting showed that ApoD protein was present in the head, hemolymph, fat body, gonad, middle silk gland, and posterior silk gland. ApoD expression was relatively higher in the hemolymph during days 1–3 of fifth-instar larvae and days 6–8 of the pupal stage. The highest mRNA levels were detected in the pupae on day 9 by using quantitative RT-PCR. After an oxidative stress treatment and a bacterial challenge, expression levels of the ApoD gene from B. mori were up-regulated at different time points compared with the control group. Simultaneously, immunohistochemical localization showed that ApoD is widely detected in the brain, eye, ovary, and testis on day 3 of the fifth-instar larval stage. The results indicated that ApoD is involved in insect development and reproduction, and plays an important role in the response to bacterial challenge and other stress signals.

Apolipoprotein D deficiency is associated to high bone turnover, low bone mass and impaired osteoblastic function in aged female mice

BackgroundApolipoprotein D (ApoD) is a member of the lipocalin family known to transport small hydrophobic ligands. A major site of ApoD expression in mice is the central nervous system where evidence suggests that it plays a protective role. Gene expression of ApoD was reported in bone-forming osteoblasts but its impact on bone metabolism remains undocumented. MethodsWe compared basic bone parameters of ApoD−/− (null) and transgenic (tg) mice to wild-type (wt) littermates through microCT and histochemistry, as well as ApoD expression and secretion in osteoblasts under various culture conditions through real-time PCR and immunoblotting. ResultsApoD-null females displayed progressive bone loss with aging, resulting in a 50% reduction in trabecular bone volume and a 23% reduction in cortical bone volume by 9months of age. Only cortical bone volume was significantly reduced in ApoD-null males by an average of 24%. Histochemistry indicated significantly higher osteoblast surface and number of osteoclasts in femora from ApoD-null females. ApoD gene expression was confirmed in primary cultures of bone marrow mesenchymal cells (MSC), with higher expression levels in MSC from females compared to males. ApoD-null MSC exhibited impaired proliferation and differentiation potentials. Moreover, exogenous ApoD partially rescued the osteogenic potential of null MSC, which were shown to readily uptake the protein from media. ApoD expression was upregulated under low proliferation conditions, by contact inhibition and osteoblastic differentiation in MC3T3-E1 osteoblast-like cells. ConclusionOur results indicate that ApoD influences bone metabolism in mice in a gender-specific manner, potentially through an auto-/paracrine pathway.

Binding and Repressive Activities of Apolipoprotein E3 and E4 Isoforms on the Human ApoD Promoter

Apolipoprotein D (ApoD) gene expression is increased in several neurological disorders such as Alzheimer’s disease (AD) and multiple sclerosis. We previously showed that transgenic mice that overexpress human ApoD show a better resistance against paraquat or OC43 coronavirus-induced neurodegeneration. Here, we identified several nuclear factors from the cortex of control and OC43-infected mice which bind a fragment of the proximal ApoD promoter in vitro. Of interest, we detected apolipoprotein E (ApoE). Human ApoE consists of three isoforms (E2, E3, and E4) with the E4 and E2 alleles representing a greater and a lower risk for developping AD, respectively. Our results show that ApoE is located in the nucleus and on the ApoD promoter in human hepatic and glioblastoma cells lines. Furthermore, overexpression of ApoE3 and ApoE4 isoforms but not ApoE2 significantly inhibited the ApoD promoter activity in U87 cells (E3/E3 genotype) cultured under normal or different stress conditions while ApoE knock-down by siRNA had a converse effect. Consistent with these results, we also demonstrated by ChIP assay that E3 and E4 isoforms, but not E2, bind the ApoD promoter. Moreover, using the Allen Brain Atlas in situ hybridization database, we observed an inverse correlation between ApoD and ApoE mRNA expression during development and in several regions of the mouse brain, notably in the cortex, hippocampus, plexus choroid, and cerebellum. This negative correlation was also observed for cortex layers IV–VI based on a new Transcriptomic Atlas of the Mouse Neocortical Layers. These findings reveal a new function for ApoE by regulating ApoD gene expression.Electronic supplementary materialThe online version of this article (doi:10.1007/s12035-013-8456-0) contains supplementary material, which is available to authorized users.

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 2 h. Altogether, these findings demonstrate the importance of Parp-1, APEX-1 and ERK1/2 catalytic activities in the growth arrest-induced apoD gene expression.

Downregulation of intracellular apolipoprotein D in DNA-PKcs-independent V(D)J recombination (36.16)

Abstract Recombination-inducible mouse pre-B cell lines are models for intracellular events that occur during lymphocyte V(D)J recombination. Using the ts-abl transformed pre-B cell lines with wild type DNA-PKcs or lines from SCID mice that have a DNA-PKcs deficiency, they can be induced to recombine their V(D)J genes segments. One of the events coincident with recombination is G1 cell cycle arrest that is much more dramatic in the SCID pre-B cells. We have recently become interested in intracellular apolipoprotein D (ApoD), an ancient intracellular lipocalin. ApoD has been described to have a role in extending lifespan by modulating cellular stress responses. ApoD would be expected to be upregulated in cells undergoing cell cycle arrest as this is a potentially stressful event for dividing cells. However, when assessing the effects of cell cycle arrest in the pre-B cell lines, we found that the intracellular Apo D actually decreases. The SCID pre-B cells, having a more severe G1 cycle arrest from recombination induction, also show a more dramatic reduction in ApoD levels. It is unclear whether the down-regulation of ApoD is associated with the recombination induction or the cell cycle arrest events. Since the SCID pre-B cells are deficient in DNA-PKcs function, it is tempting to speculate that ApoD gene expression may be influenced by this protein kinase. Nevertheless, these data suggest a previously undescribed role for intracellular ApoD in B-cell V(D)J recombination events.

Modulation of Apolipoprotein D and Apolipoprotein E mRNA Expression by Growth Arrest and Identification of Key Elements in the Promoter

Apolipoprotein D (apoD) and apolipoprotein E (apoE) are co-expressed in many tissues, and, in certain neuropathological situations, their expression appears to be under coordinate regulation. We have previously shown that apoD gene expression in cultured human fibroblasts is up-regulated when the cells undergo growth arrest. Here, we demonstrate that, starting around day 2 of growth arrest, both apoD and apoE mRNA levels increase between 1.5- and 27-fold in other cell types, including mouse primary fibroblasts and fibroblast-like and human astrocytoma cell lines. To understand the regulatory mechanisms of apoD expression, we have used apoD promoter-luciferase reporter constructs to compare gene expression in growing cells and in cells that have undergone growth arrest. Analysis of gene expression in cells transfected with constructs with deletions and mutations in the apoD promoter and constructs with artificial promoters demonstrated that the region between nucleotides -174 and -4 is fully responsible for the basal gene expression, whereas the region from -558 to -179 is implicated in the induction of apoD expression following growth arrest. Within this region, an alternating purine-pyrimidine stretch and a pair of serum-responsive elements (SRE) were found to be major determinants of growth arrest-induced apoD gene expression. Evidence is also presented that SREs in the apoE promoter may contribute to the up-regulation of apoE gene expression following growth arrest.

Cellular cholesterol storage in the Niemann-Pick disease type C mouse is associated with increased expression and defective processing of apolipoprotein D.

Apolipoprotein D (apoD), a member of the lipocalin superfamily of ligand transporters, has been implicated in the transport of several small hydrophobic molecules including sterols and steroid hormones. We have previously established that apoD is a secreted protein from cultured mouse astrocytes and that treatment with the oxysterol 25-hydroxycholesterol markedly stimulates apoD release. Here, we have investigated expression and cellular processing of apoD in the Niemann-Pick type C (NPC) mouse, an animal model of human NPC, which is a genetic disorder affecting cellular cholesterol transport. NPC is phenotypically characterized by symptoms of chronic progressive neurodegeneration. ApoD gene expression was up-regulated in cultured NPC astrocytes and in NPC brain. ApoD protein levels were also increased in NPC brain with up to 30-fold higher apoD content in the NPC cerebellum compared with control mice. Subcellular fractionation of NPC brain homogenates revealed that most of the apoD was associated with the myelin fraction. ApoD was found to be a secreted protein from cultured normal astrocytes and treatment with the oxysterol, 25-hydroxycholesterol, markedly stimulated apoD release (by five- to 10-fold). By contrast, secretion of apoD from NPC astrocytes was markedly reduced and could not be stimulated by oxysterol treatment. Secretion of apoE, another apolipoprotein normally produced by astrocytes, was similar in NPC and control cells. Furthermore, apoE secretion was not potentiated by oxysterol treatment in either cell type. Plasma levels of apoD were sixfold higher in NPC, whereas hepatic levels were substantially reduced compared with controls, possibly reflecting reduced hepatic clearance of the circulating protein. These results reveal hitherto unrecognized defects in apoD metabolism in NPC that appear to be linked to the known defects in cholesterol homeostasis in this disorder.

Retinoic Acid-induced Expression of Apolipoprotein D and Concomitant Growth Arrest in Human Breast Cancer Cells Are Mediated through a Retinoic Acid Receptor RARα-dependent Signaling Pathway

Apolipoprotein D (apoD) is a human plasma protein, belonging to the lipocalin superfamily, that is produced by a specific subtype of highly differentiated breast carcinomas and that is strongly up-regulated by retinoic acid (RA) in breast cancer cells. In this work, we have examined the molecular mechanisms mediating the induction of apoD gene expression by retinoids in T-47D human breast cancer cells. Northern blot analysis revealed that Ro40-6055, a synthetic retinoid that selectively binds and activates the retinoic acid receptor RARalpha, induced the accumulation of apoD mRNA in breast cancer cells in a time- and dose-dependent manner. The time course analysis demonstrated that apoD mRNA was induced 14-fold over control cells after 48 h of incubation with 10(-8) M Ro40-6055. As little as 10(-11) M of this retinoid induced apoD mRNA 5-fold over the control, whereas incubation with 10(-7) M Ro40-6055 induced maximally 15-fold over control cells. RARalpha-selective antagonists counteracted the inductive effects of all-trans-RA, 9-cis-RA, and Ro40-6055 on the expression of apoD, when present at the same concentration as the retinoid agonists. By contrast, RARbeta-, RARgamma-, and RXR-selective retinoids did not affect apoD gene expression. The retinoid agonist Ro40-6055 had an antiproliferative effect on T-47D cells, with maximal growth inhibition of approximately 60% obtained after 7 days of incubation with 10(-7) M. This antiproliferative effect could be counteracted by a 100-fold excess of the antagonist Ro41-5253. Treatment of the cells with retinoids that do not bind the nuclear retinoic acid receptors did not affect apoD expression, despite the fact that they did have a strong antiproliferative effect on T-47D cells. On the basis of these results, a role for RARalpha on apoD gene expression induction by retinoids in breast cancer cells is proposed.

Guinea pig apolipoprotein D RNA diversity, and developmental and gestational modulation of mRNA levels

We have isolated and characterized two molecular types of guinea pig (GP) apolipoprotein D (apoD) cDNA. The sequences of cDNA clones GP APO D-20 and -38 are 100% homologous in their putative exons 2–5, as determined by analogy with human apoD gene, but they differ totally in their putative exon 1. RNase protection assays showed the presence of both apoD RNA types 20 and 38 in cauda epididymis. Northern blot analysis revealed four polyadenylated apoD bands at 3.2, 2.7, 1.7, and 1.0 kb. Types 20 and 38 specific probes hybridized with the major 1-kb mRNA and two of the three other minor RNA transcripts, respectively. Southern blot analysis revealed that the guinea pig genome probably contains one apoD gene. Our data also demonstrated that the cauda epididymis and fallopian tubes had an apoD mRNA concentration 100-fold higher than the liver, suggesting that the apoD gene expression could be associated with the presence of steroids. The levels of the 1-kb mRNA increased in the fallopian tubes and ovaries during gestation and were lower in fetal reproductive tissues and liver than in mature animals. No positive correlation was found between apoD and 3β-hydroxysteroid dehydrogenase/ Δ 5- Δ 4 isomerase (3β-HSD) mRNA levels in these tissues, thus suggesting that high amounts of apoD mRNA are not necessarily associated with in situ progesterone synthesis. Taken together, our results indicate that both the guinea pig epididymis and fallopian tubes are excellent models to study the local role of apoD in steroid target tissues.

Structure of the human apolipoprotein D gene promoter region

A human genomic clone of 18.2 kbp encompassing the apolipoprotein D (apoD) exon-1 and 2 and 10 kbp of upstream sequence was isolated and characterized. DNA sequencing and primer extension analysis revealed a transcriptional initiation site located 27 bp downstream of a consensus TATA box sequence. The exon 1 was 66 bp long. Computer analysis of DNA sequence from positions −557 to +129 revealed some putative transcriptional regulatory elements including a stretch of (pyrimidine/purine) 26 located from nucleotide −263 to nucleotide −212, which could potentially form Z-DNA. Steroid hormone regulatory elements were identified which may be related to the modulation of apoD gene expression by androgens and estrogens in vitro.

Additive stimulatory action of glucocorticoids and androgens on basal and estrogen-repressed apolipoprotein-D messenger ribonucleic acid levels and secretion in human breast cancer cells.

Recent elucidation of the amino acid sequence of the progesterone-binding protein GCDFP-24, the major protein found in human breast gross cystic disease fluid, reveals that this glycoprotein corresponds to apolipoprotein-D (apo-D), a member of the alpha 2-microglobulin superfamily which binds small hydrophobic ligands. The present study describes the multiple hormonal control of apo-D mRNA levels, intracellular protein content, as well as secretion compared to cell proliferation in human ZR-75-1 breast cancer cells. In these cells, exposure to the synthetic glucocorticoid dexamethasone (DEX) markedly decreases basal as well as 17 beta-estradiol (E2)-induced cell proliferation while causing a maximal 10-fold stimulation of apo-D secretion in the presence or absence of E2. Incubation with 500 nM DEX or 10 nM dihydrotestosterone (DHT), alone and in combination, markedly increased apo-D mRNA/actin mRNA ratios by 16-, 22-, and 28-fold, respectively. Exposure to 1 nM E2 decreased the apo-D mRNA/actin mRNA ratio by 65%. In E2-treated cells, simultaneous exposure to DHT, DEX, and DHT plus DEX markedly increased the apo-D mRNA/actin mRNA ratios by 50-, 35-, and 105-fold, respectively. The stimulatory effect of DEX on intracellular apo-D content and secretion was also additive to that of the androgen DHT in the presence or absence of E2. The present study provides the first data describing the hormonal regulation of apo-D mRNA levels and intracellular protein content and demonstrates the effect of glucocorticoids alone as well as their interaction with androgens and estrogens on these parameters as well as on apo-D secretion. As shown in the present data, the effects of steroids on apo-D gene expression, intracellular apo-D protein content, and secretion are opposite their respective specific effects on cell proliferation in human ZR-75-1 breast cancer cells.