ApoD Expression Research Articles

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.

Apolipoprotein D mRNA expression is elevated in PDAPP transgenic mice.

Apolipoprotein D (apoD) expression is known to be elevated in select regions of rodent and human brain in association with different types of CNS pathology. To investigate a potential role for apoD in the neuropathology of Alzheimer's disease, we have measured apoD mRNA expression in transgenic mice expressing mutated human amyloid precursor protein under control of platelet-derived growth factor promoter (PDAPP mice). In situ hybridization analysis revealed increased apoD mRNA expression in brains of aged (26 months) PDAPP transgenic mice compared to aged littermate controls. These increases were most prominent in the hippocampal fimbria, corpus callosum and other white matter tracts. No substantial increases in expression were observed in white matter regions in young (6 months) PDAPP transgenic mice compared to young controls. Comparison between aged and young control mice revealed increased apoD expression in similar white matter regions of the aged animals. These findings suggest that, although increases in apoD expression are a normal feature of brain aging, super-increases may represent a glial cell compensatory response to beta-amyloid deposition in Alzheimer's disease.

Expression pattern of the lipocalin apolipoprotein D during mouse embryogenesis.

Apolipoprotein D (ApoD) is a secreted protein that belongs to the lipocalin family. We describe the expression pattern of ApoD during mouse embryogenesis by in situ hybridization using RNA probes. ApoD is expressed at E9 in mesenchymal cells in the rombencephalic-mesencephalic region. At E9.5 the cephalic ApoD-positive cells appear in the mesenchyme, and at later stages (starting at E10.5) ApoD expression is seen in meninges. Within the neuroepithelium, ApoD is expressed in pericytes surrounding brain and spinal cord capillaries from E10.5 to birth. Other places of expression of ApoD are the mesenchyme surrounding the olfactory epithelium and semicircular canals, as well as chondroblasts of skull and vertebrae.

Increased CNS levels of apolipoprotein D in schizophrenic and bipolar subjects: implications for the pathophysiology of psychiatric disorders.

Chronic administration of the atypical antipsychotic drug, clozapine, to rodents has been shown to increase the concentration of apolipoprotein D (apoD) in several area of the brain, suggesting that apoD could be involved in the therapeutic effects of antipsychotic drugs and/or the pathology of psychotic illnesses. Here, we measured a significant decrease in the concentration of apoD in serum samples from schizophrenic patients. In contrast, apoD levels were significantly increased (92--287%) in dorsolateral prefrontal cortex (Brodmann's area 9) of schizophrenic and bipolar subjects. Elevated levels of apoD expression were also observed in the caudate of schizophrenic and bipolar subjects (68--89%). No differences in apoD immunoreactivity were detected in occipital cortex (Brodmann's area 18) in either group, or in the hippocampus, substantia nigra, or cerebellum of the schizophrenic group. The low serum concentrations of apoD observed in these patients supports recent hypotheses involving systemic insufficiencies in lipid metabolism/signaling in schizophrenia. Elevation of apoD expression selectively within central nervous system regions implicated in the pathology of these neuropsychiatric disorders suggests a focal compensatory response that neuroleptic drug regimens may augment.

Clozapine increases apolipoprotein D expression in rodent brain: towards a mechanism for neuroleptic pharmacotherapy.

In contrast to typical neuroleptic drugs, which have high affinities for dopamine D2 receptors, clozapine binds to multiple neurotransmitter receptors. The mechanisms responsible for its superior clinical efficacy over typical neuroleptics remain unknown. Using an automated genomics approach, total gene expression analysis (TOGA), we found an approximately threefold increase in the accumulation of the mRNA encoding apolipoprotein D (apoD) in mouse striatum in response to chronic treatment with clozapine. While in control animals, apoD is expressed predominantly in astrocytes, in situ hybridization and immunohistochemical studies indicated a substantial increase in apoD expression in neurons of the striatum, globus pallidus and thalamus after 2 weeks of clozapine treatment. Clozapine-induced increases in apoD expression were also observed in some white matter regions. These results suggest that apoD is a mediator in the mechanisms of clozapine and thus that deficiencies in aspects of lipid metabolism may be responsible for psychoses.

Immunocytochemical localization of apolipoprotein D in oligodendrocyte precursor-like cells, perivascular cells, and pericytes in the human cerebral cortex.

Apolipoprotein D, a lipocalin transporter of small hydrophobic molecules including sterols, steroid hormones and arachidonic acid, is a widely expressed protein in peripheral and neural tissues. It has been shown to be upregulated in the context of neural injury, and with neuronal degeneration and regeneration. Here we have used light and electron microscopic immunocytochemistry with immunogold labeling to delineate the pattern of expression of apoD in the human brain. Our results confirm previous observations that apoD is a predominantly glial protein in the nervous system. In addition we have found that apoD is present in the cytosol and outer membrane of the nuclear envelope of glial cells in the neuropil. The labeled glial cells were putatively identified as a population of oligodendrocyte precursor cells. Immunoreactivity was also associated with the cytosol of perivascular cells, and lysosomes of pericytes, in the walls of blood vessels. These observations suggest a potential role for glial cells and apoD, in the transport of sterols and small hydrophobic molecules to, or from, blood vessels in the cortex.

Increased expression of apolipoprotein D following experimental traumatic brain injury.

Increasing evidence suggests that apolipoprotein D (apoD) could play a major role in mediating neuronal degeneration and regeneration in the CNS and the PNS. To investigate further the temporal pattern of apoD expression after experimental traumatic brain injury in the rat, male Sprague-Dawley rats were subjected to unilateral cortical impact injury. The animals were killed and examined for apoD mRNA and protein expression and for immunohistological analysis at intervals from 15 min to 14 days after injury. Increased apoD mRNA and protein levels were seen in the cortex and hippocampus ipsilateral to the injury site from 48 h to 14 days after the trauma. Immunohistological investigation demonstrated a differential pattern of apoD expression in the cortex and hippocampus, respectively: Increased apoD immunoreactivity in glial cells was detected from 2 to 3 days after the injury in cortex and hippocampus. In contrast, increased expression of apoD was seen in cortical and hippocampal neurons at later time points following impact injury. Concurrent histopathological examination using hematoxylin and eosin demonstrated dark, shrunken neurons in the cortex ipsilateral to the injury site. In contrast, no evidence of cell death was observed in the hippocampus ipsilateral to the injury site up to 14 days after the trauma. No evidence of increased apoD mRNA or protein expression or neuronal pathology by hematoxylin and eosin staining was detected in the contralateral cortex and hippocampus. Our results reveal induction of apoD expression in the cortex and hippocampus following traumatic brain injury in the rat. Our data also suggest that increased apoD expression may play an important role in cortical neuronal degeneration after brain injury in vivo. However, increased expression of apoD in the hippocampus may not necessarily be indicative of neuronal death.

Modulation of apolipoprotein D and apolipoprotein E expression in rat hippocampus after entorhinal cortex lesion

Apolipoprotein (apo) D is a member of the lipocalin family of proteins. Although its physiological function is unknown, apoD is thought to transport one or more small hydrophobic ligands. A second apolipoprotein, apoE is known to play an important role in lipid transport, and apoE genetic polymorphism has been shown to be associated with susceptibility to Alzheimer's disease. Both apoD and apoE are expressed in the central nervous system (CNS) and both proteins accumulate at sites of peripheral nerve injury due to increased local synthesis. The two proteins may have overlapping or complementary functions within nervous tissue. In order to define the role of apoD within the CNS, we have studied the regional distribution of apoD and apoE mRNA and protein within the normal rat brain and the changes in apoD and apoE expression in the hippocampus of rats after entorhinal cortex lesion (EC lesion). Within the brains of normal rats, apoD expression in the hippocampus was as high as 180-fold that of the liver. ApoD mRNA levels in other areas of the rat brain ranged from 40 to 120 times the hepatic levels. The distribution of apoE gene expression within the brain was similar to that of apoD, but was much lower than hepatic apoE expression. When rats were subjected to EC lesion, the apoD message increased by 54% at 4 days post lesion (DPL) in the ipsilateral region of hippocampus while apoE mRNA levels (ipsilateral and contralateral) decreased by 43%. At 6 to 8 DPL apoD mRNA in the ipsilateral hippocampus remained elevated (42% above controls) whereas the apoE mRNA levels increased to about 15% above those of controls. At 14 and 31 DPL, both apoD and apoE expression was similar to controls. The increase in immunoreactive apoD in hippocampal extracts was more dramatic. At 1 DPL, immunoreactive apoD levels were already 16-fold higher than those in extracts of non-lesioned animals and, at 31 DPL, levels were still 8-fold higher than those of control animals. Finally, we have demonstrated that the levels of apoD in the brains of apoE-deficient mice are 50-fold those of wildtype control mice. ApoD clearly has an important function within the CNS in both normal and pathological situations.

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.

Molecular cloning of the mouse apolipoprotein D gene and its upregulated expression in Niemann-Pick disease type C mouse model.

Apolipoprotein D (ApoD) is a member of the lipocalin superfamily. The primary structure and diverse expression of ApoD suggest that this protein is a multiligand, multifunctional glycoprotein. Here we report the structure of the mouse ApoD gene, which is composed of six exons spanning approximately 20 kb in length. All the exon-intron splice junctions follow the consensus GT/AG sequence. The 5'-flanking region of the mouse ApoD gene contains several putative regulatory elements, including FSE-2, GRE, SDR, MRE, IL-6RE, and TATA box. Northern blot analysis revealed that ApoD was highly expressed in the brain and adipose tissue in mouse. Lower levels of expression were observed in the heart, lung, thymus, testis, and salivary glands. In situ hybridization for the brain showed that ApoD mRNA was mainly localized in the subarachnoid space including the pia. In the Niemann-Pick disease type C mouse model, ApoD expression was upregulated in many organs such as brain, adipose tissue, heart, and thymus, presumably due to enhanced ApoD synthesis in perivascular fibroblasts.

Expression of the androgen receptor and an androgen-responsive protein, apolipoprotein D, in human breast cancer.

Little is known regarding the activity and function of the androgen receptor (AR) in human breast cancer. In the present study AR was evaluated in untreated primary breast cancers using antisera to the amino- and carboxy-termini of the receptor and quantitated using colour video image analysis. A strong correlation between tissue concentration and percentage AR-positive cells was observed for each antiserum. However, comparison of percentage positive cells using the amino- and carboxy-terminal AR antisera in individual breast cancer specimens revealed a subset of tumours with discordantly increased staining for the carboxy terminus. These findings suggest the presence of amino-terminal-truncated AR in a proportion of breast cancer cells or presence of AR mutations or associated protein alterations that affect binding of the amino-terminal AR antiserum. Immunohistochemical expression of the androgen-regulated glycoprotein, apolipoprotein D (apo-D), was also evaluated in the breast cancer specimens. Focal positivity of apo-D staining, which did not always co-localise with AR-positive cells, was observed within breast tumours. Furthermore, no correlation was evident between percentage positive cells stained for AR and apo-D in breast cancer specimens. These findings indicate that, although apo-D expression is androgen regulated in human breast cancer cell lines in vitro, its expression in primary breast cancers may be regulated by other factors. The expression of AR in primary breast cancers also suggests that the receptor may be involved in tumour responsiveness or in abnormal responses to endocrine therapies.

Inverse relationships between cell proliferation and basal or androgen-stimulated apolipoprotein D secretion in LNCaP human prostate cancer cells

We have recently demonstrated that the biphasic action of androgens on LNCaP cell proliferation is opposite to their effect on apolipoprotein D (apo-D) secretion, the stimulation of apo-D secretion being associated with a steroid-induced inhibition of cell proliferation. To further characterize the control of apo-D expression in LNCaP cells, we studied basal as well as androgen-induced apo-D secretion in slowly proliferating, low-passage (LP; 20–29th) and rapidly proliferating high-passage (HP; 111–117th) cell cultures. For comparison, the androgen-induced stimulation of prostate specific antigen secretion was also investigated in LP and HP cell cultures. In the absence of androgens, basal cell proliferation of HP cells was significantly higher than that of LP cells, whereas apo-D secretion was higher in LP cells than in HP cells. Furthermore, the biphasic action of dihydrotestosterone and of the synthetic androgenic compound R1881 on apo-D release and cell proliferation was observed in both LP and HP cells. The stimulation of apo-D secretion was inversely related to that of cell proliferation and influenced by cell density. The inhibition of basal and androgen-induced cell proliferation by the calcium channel blocker nifedipine was also associated with an increase in apo-D secretion. The amount of PSA released and the sensitivity of its response to R1881 were increased in LP cells compared with HP cells. The present study thus demonstrates, for the first time, that apo-D secretion is inversely correlated to cell proliferation and cell density in the absence as well as in the presence of androgens in both LP and HP LNCaP human prostate cancer cells. This finding suggests that apo-D expression can be modulated not only by steroid hormones, but also by other factors involved in the control of cell proliferation.

Apolipoprotein D gene induction by retinoic acid is concomitant with growth arrest and cell differentiation in human breast cancer cells.

We have examined the regulation by retinoic acid of the gene encoding apolipoprotein D (apoD), a human plasma protein belonging to the superfamily of the lipocalins that is produced by a specific subtype of highly differentiated breast carcinomas. Northern blot analysis revealed that all-trans-retinoic acid (RA) strongly induced the accumulation of apoD mRNA in T-47D and ZR-75-1 estrogen receptor-positive human breast cancer cells in a time- and dose-dependent manner, while no inductive effect was observed in estrogen receptor-negative cell lines, including MDA-MB-231 and MDA-MB-435. The effect of RA on apoD expression by T-47D cells was at least 12-fold more potent than the effect of the steroids dihydrotestosterone and dexamethasone, which had been previously described as hormonal up-regulators of apoD expression in these cells. A time course study demonstrated that the induction of apoD mRNA reached a level of 15-fold over the untreated control cells after 48 h of incubation in the presence of a 10(-7) M concentration of RA. A dose-response analysis showed that as little as 10(-13) M RA produced an accumulation of 5-fold over the control, while incubation of the cells in the presence of 10(-5) M RA induced a maximal accumulation of 24-fold over the control untreated cells. The induction of apoD mRNA was independent of the synthesis of proteins de novo, as demonstrated by the fact that the induction was also detected in the presence of cycloheximide. The incubation of the cells in the presence of RA did not affect significantly the stability of apoD mRNA. By contrast, treatment of the T-47D cells with RA produced an increase of approximately 8-fold in the rate of transcription of the apoD gene. Furthermore, treatment of the T-47D cells with RA induced the synthesis and secretion to the culture medium of apoD. This increased expression of apoD was accompanied by an inhibition of cell proliferation and a progression through a more differentiated phenotype, suggesting that the mechanisms controlling RA-induced growth arrest, cell differentiation, and apoD synthesis may be directly coordinated in human breast cancer cells.

Apolipoprotein D transcription occurs specifically in nonproliferating quiescent and senescent fibroblast cultures

We studied apolipoprotein D (apoD) mRNA in primary cultures of human diploid fibroblasts (HDF). In early-passage HDF no apoD mRNA was detected in replicating cells in sparse culture, but the gene was expressed in quiescent cells in confluent and in serum-starved cultures. In contrast, late-passage HDF expressed apoD mRNA in sparse culture, but the level increased after attainment of confluence. Thus fibroblasts, the common cell-type expressing apoD mRNA in vivo, express this characteristic following growth-arrest. The same pattern of activation was found in another fibroblast cell line deficient in apoB/E (LDL) receptors, excluding a role for cellular cholesterol delivery by the LDL-receptor pathway controlling apoD expression