Apolipoprotein E - A Multifunctional Protein with Implications in Various Pathologies as a Result of Its Structural Features

Apolipoprotein E (apoE), a 34 kDa glycoprotein, mediates hepatic and extrahepatic uptake of plasma lipoproteins and cholesterol efflux from lipid-laden macrophages. In humans, three structural different apoE isoforms occur, with subsequent functional changes and pathological consequences. Here, we review data supporting the involvement of apoE structural domains and isoforms in normal and altered lipid metabolism, cardiovascular and neurodegenerative diseases, as well as stress-related pathological states. Studies using truncated apoE forms provided valuable information regarding the regions and residues responsible for its properties. ApoE3 renders protection against cardiovascular diseases by maintaining lipid homeostasis, while apoE2 is associated with dysbetalipoproteinemia. ApoE4 is a recognized risk factor for Alzheimer's disease, although the exact mechanism of the disease initiation and progression is not entirely elucidated. ApoE is also implicated in infections with herpes simplex type-1, hepatitis C and human immunodeficiency viruses. Interacting with both viral and host molecules, apoE isoforms differently interfere with the viral life cycle. ApoE exerts anti-inflammatory effects, switching macrophage phenotype from the proinflammatory M1 to the anti-inflammatory M2, suppressing CD4+ and CD8+ lymphocytes, and reducing IL-2 production. The anti-oxidative properties of apoE are isoform-dependent, modulating the levels of various molecules (Nrf2 target genes, metallothioneins, paraoxonase). Mimetic peptides were designed to exploit apoE beneficial properties. The “structure correctors” which convert apoE4 into apoE3-like molecules have pharmacological potential. Despite no successful strategy is yet available for apoE-related disorders, several promising candidates deserve further improvement and exploitation. Keywords: ApoE, Isoform, Mimetic peptide, Structural domain, Truncated molecul

STAT1 interacts with RXRα to upregulate ApoCII gene expression in macrophages.

Apolipoprotein CII (apoCII) is a specific activator of lipoprotein lipase and plays an important role in triglyceride metabolism. The aim of our work was to elucidate the regulatory mechanisms involved in apoCII gene modulation in macrophages. Using Chromosome Conformation Capture we demonstrated that multienhancer 2 (ME.2) physically interacts with the apoCII promoter and this interaction facilitates the transcriptional enhancement of the apoCII promoter by the transcription factors bound on ME.2. We revealed that the transcription factor STAT1, previously shown to bind to its specific site on ME.2, is functional for apoCII gene upregulation. We found that siRNA-mediated inhibition of STAT1 gene expression significantly decreased the apoCII levels, while STAT1 overexpression in RAW 264.7 macrophages increased apoCII gene expression. Using transient transfections, DNA pull down and chromatin immunoprecipitation assays, we revealed a novel STAT1 binding site in the -500/-493 region of the apoCII promoter, which mediates apoCII promoter upregulation by STAT1. Interestingly, STAT1 could not exert its upregulatory effect when the RXRα/T3Rβ binding site located on the apoCII promoter was mutated, suggesting physical and functional interactions between these factors. Using GST pull-down and co-immunoprecipitation assays, we demonstrated that STAT1 physically interacts with RXRα. Taken together, these data revealed that STAT1 bound on ME.2 cooperates with RXRα located on apoCII promoter and upregulates apoCII expression only in macrophages, due to the specificity of the long-range interactions between the proximal and distal regulatory elements. Moreover, we showed for the first time that STAT1 and RXRα physically interact to exert their regulatory function

K2 Transfection System Boosts the Adenoviral Transduction of Murine Mesenchymal Stromal Cells

Adenoviral vectors are important vehicles for delivering therapeutic genes into mammalian cells. However, the yield of the adenoviral transduction of murine mesenchymal stromal cells (MSC) is low. Here, we aimed to improve the adenoviral transduction efficiency of bone marrow-derived MSC. Our data showed that among all the potential transduction boosters that we tested, the K2 Transfection System (K2TS) greatly increased the transduction efficiency. After optimization of both K2TS components, the yield of the adenoviral transduction increased from 18% to 96% for non-obese diabetic (NOD)-derived MSC, from 30% to 86% for C57BL/6-derived MSC, and from 0.6% to 63% for BALB/c-derived MSC, when 250 transduction units/cell were used. We found that MSC derived from these mouse strains expressed different levels of the coxsackievirus and adenovirus receptors (MSC from C57BL/6≥NOD>>>BALB/c). K2TS did not increase the level of the receptor expression, but desensitized the cells to foreign DNA and facilitated the virus entry into the cell. The expression of Stem cells antigen-1 (Sca-1) and 5′-nucleotidase (CD73) MSC markers, the adipogenic and osteogenic differentiation potential, and the immunosuppressive capacity were preserved after the adenoviral transduction of MSC in the presence of the K2TS. In conclusion, K2TS significantly enhanced the adenoviral transduction of MSC, without interfering with their main characteristics and properties

K2 Transfection System Boosts the Adenoviral Transduction of Murine Mesenchymal Stromal Cells

Adenoviral vectors are important vehicles for delivering therapeutic genes into mammalian cells. However, the yield of the adenoviral transduction of murine mesenchymal stromal cells (MSC) is low. Here, we aimed to improve the adenoviral transduction efficiency of bone marrow-derived MSC. Our data showed that among all the potential transduction boosters that we tested, the K2 Transfection System (K2TS) greatly increased the transduction efficiency. After optimization of both K2TS components, the yield of the adenoviral transduction increased from 18% to 96% for non-obese diabetic (NOD)-derived MSC, from 30% to 86% for C57BL/6-derived MSC, and from 0.6% to 63% for BALB/c-derived MSC, when 250 transduction units/cell were used. We found that MSC derived from these mouse strains expressed different levels of the coxsackievirus and adenovirus receptors (MSC from C57BL/6≥NOD>>>BALB/c). K2TS did not increase the level of the receptor expression, but desensitized the cells to foreign DNA and facilitated the virus entry into the cell. The expression of Stem cells antigen-1 (Sca-1) and 5′-nucleotidase (CD73) MSC markers, the adipogenic and osteogenic differentiation potential, and the immunosuppressive capacity were preserved after the adenoviral transduction of MSC in the presence of the K2TS. In conclusion, K2TS significantly enhanced the adenoviral transduction of MSC, without interfering with their main characteristics and properties

Differential action of glucocorticoids on apolipoprotein E gene expression in macrophages and hepatocytes.

Apolipoprotein E (apoE) has anti-atherosclerotic properties, being involved in the transport and clearance of cholesterol-rich lipoproteins as well as in cholesterol efflux from cells. We hypothesized that glucocorticoids may exert anti-inflammatory properties by increasing the level of macrophage-derived apoE. Our data showed that glucocorticoids increased apoE expression in macrophages in vitro as well as in vivo. Dexamethasone increased ~6 fold apoE mRNA levels in cultured peritoneal macrophages and RAW 264.7 cells. Administered to C57BL/6J mice, dexamethasone induced a two-fold increase in apoE expression in peritoneal macrophages. By contrast, glucocorticoids did not influence apoE expression in hepatocytes, in vitro and in vivo. Moreover, dexamethasone enhanced apoE promoter transcriptional activity in RAW 264.7 macrophages, but not in HepG2 cells, as tested by transient transfections. Analysis of apoE proximal promoter deletion mutants, complemented by protein-DNA interaction assays demonstrated the functionality of a putative glucocorticoid receptors (GR) binding site predicted by in silico analysis in the -111/-104 region of the human apoE promoter. In hepatocytes, GR can bind to their specific site within apoE promoter but are not able to modulate the gene expression. The modulatory blockade in hepatocytes is a consequence of partial involvement of transcription factors and other signaling molecules activated through MEK1/2 and PLA2/PLC pathways. In conclusion, our study indicates that glucocorticoids (1) differentially target apoE gene expression; (2) induce a significant increase in apoE level specifically in macrophages. The local increase of apoE gene expression in macrophages at the level of the atheromatous plaque may have therapeutic implications in atherosclerosis

Adenovirus-Mediated FasL Minigene Transfer Endows Transduced Cells with Killer Potential

Fas ligand (First apoptosis signal ligand, FasL, also known as CD95L) is the common executioner of apoptosis within the tumor necrosis factor (TNF) superfamily. We aimed to induce functional FasL expression in transduced cells using an adenovirus vector, which has the advantage of strong and transient induction of the gene included in the adenoviral genome. Here, we report that the adenovirus carrying a truncated FasL gene, named FasL minigene, encoding the full-length FasL protein (Ad-gFasL) is more efficient than the adenovirus carrying FasL cDNA (Ad-cFasL) in the induction of FasL expression in transduced cells. FasL minigene (2887 bp) lacking the second intron and a part of the 3′-UTR was created to reduce the gene length due to the size limitation of the adenoviral genome. The results show that, in transduced hepatocytes, strong expression of mRNA FasL appeared after 10 h for Ad-gFasL, while for Ad-cFasL, a faint expression appeared after 16 h. For Ad-gFasL, the protein expression was noticed starting with 0.5 transfection units (TU)/cell, while for Ad-cFasL, it could not be revealed. FasL-expressing endothelial cells induced apoptosis of A20 cells in co-culture experiments. FasL-expressing cells may be exploitable in various autoimmune diseases such as graft-versus-host disease, chronic colitis, and type I diabetes

Transactivation of apoCII promoter by STAT1 is amplified by RXRα ligand in macrophages, but not in hepatocytes.

<p><i>Panel A – D.</i> RAW 264.7 macrophages and HepG2 hepatocytes were transiently transfected with the constructs [−545/+18]apoCII-luc, [−545/+18]apoCIImut-luc, ME.2[−388/+18]apoCII-luc, and ME.2[−388/+18]apoCIImut-luc under basal condition (control), in the presence of STAT1 overexpression (STAT1), after 9-cis-retinoic acid treatment (RA) or in the presence of both activators (STAT1+RA). The concomitant STAT1 overexpression and RA treatment of RAW 264.7 macrophages enhanced the wild type apoCII promoter at a higher level than each activator alone (Panel A, grey columns). This effect on apoCII promoter was more pronounced when the promoter was under the control of ME.2 (Panel C, grey columns). The activity of the mutant apoCII promoter was not affected by either activator alone (STAT1 or RA) or in combination (Panel B, grey columns). In the presence of ME.2, RA treatment of RAW 264.7 cells increased the activity of the mutant apoCII promoter (due to the RXR binding sites found on ME.2), but STAT1 could not potentiate this enhancement (Panel D, grey columns). By contrast, in HepG2 cells the cumulative effect of STAT1 and RXR was not observed on any constructs employed (white columns).</p

STAT1-dependent transactivation of apoCII promoter in RAW 264.7 cells.

<p><i>Panel A, B.</i> RAW 264.7 macrophages (A) and HepG2 hepatocytes (B) were transiently transfected with plasmids containing [−545/+18] or [−388/+18]apoCII promoter fragments in the presence or in the absence (control) of STAT1 expression vector. STAT1 overexpression increased the activity of [−545/+18]apoCII promoter in RAW 264.7 cells (∼3.4 fold, p<0.0001) as well as in HepG2 cells (∼1.5 fold, p<0.002). By contrast, the activity of the [−388/+18]apoCII promoter was not increased by STAT1 overexpression, either in macrophages (p>0.5) or in hepatocytes. <i>Panel C.</i> RAW 264.7 macrophages were transfected with ME.2[−545/+18]apoCII-luc or with ME.2[−388/+18]apoCII-luc plasmids in the absence (control) or in the presence of STAT1 expression vector. In the presence of ME.2, the activities of [−545/+18] and [−388/+18] promoter fragments were increased by STAT1 overexpression (3.8 fold, p<0.0002 and 2.9 fold, respectively p<0.0002). <i>Panel D.</i> DNA pull down assays were performed using different fragments ([−545/+18] and [−388/+18]) of apoCII promoter or oligonucleotides containing the wild type or mutated [−507/−485] region of apoCII promoter, and nuclear extracts obtained from STAT1- overexpressing HepG2 (right) and RAW 264.7 (left) cells. The results showed that the [−545/+18]apoCII promoter, but not [−388/+18] fragment can bind STAT1 proteins (lane 1 and, respectively 2). STAT1 is able to bind the wild type [−507/−485] region of apoCII promoter (lane 4), while STAT1 binding to the mutated region is impaired (lane 5). Negative controls, in which DNAP was performed without DNA, are illustrated in lanes 3 and 6 (no DNA). <i>Panel E.</i> ChIP experiments performed using anti-STAT1 antibodies showed that STAT1 is recruited to the [−738/−336]apoCII gene fragment as well as to the [140–440] ME.2 region (lanes 1 and, respectively, 4). No bands were obtained when anti-STAT1 antibodies were omitted in ChIP experiments for apoCII promoter and for ME.2 region (lanes 2 and, respectively, 5). PCR using the input as template and primers for apoCII promoter or ME.2 gave the expected bands (lanes 3 and, respectively, 6). <i>Panel F.</i> Schematic representation of STAT1 binding sites on apoCII proximal promoter and ME.2 region.</p

Interactions between apoCII promoter and ME.2 in macrophages as revealed by chromosome conformation capture (3C) assays.

<p><i>Panels A–C.</i> Schematic illustration of the linear alignment of ME.2 and apoCII (including the Pst I sites adjacent to these regions) and the possible interactions between apoCII promoter and ME.2 in sense (B) or antisense (C) orientation. For 3C experiments, the PstI restriction enzyme was used. PstI restriction sites are located at positions +1427 in apoCII gene as well as at +677 bp downstream of ME.2 and at −1004 bp upstream of ME.2, as showed in the picture. The primers flanking the newly ligated fragments are also illustrated: for both cases (B and C), the forward primer was +1378CII, and the reverse primers were: +577ME.2 (B) and −712ME.2 (C). The predicted lengths of the ligated fragments are: 149 bp for “sense orientation” (B) and 341 bp and “antisense orientation” interaction with ME.2 (C). <i>Panel D.</i> Human peripheral blood monocytes (lanes 1–3) and THP-1 cells (lanes 4–6) differentiated to macrophages by PMA treatment, as well as HepG2 cells (lanes 7–9), were subjected to the 3C assay. PCR fragments amplified with primers +1378CII and +577ME.2 (testing the interaction in sense orientation) are shown in lanes 1, 4 and 7, and PCR products obtained with primers +1378CII and −712ME.2 (testing the interaction in antisense orientation) are shown in lanes 2, 5 and, respectively 8. The positive control for DNA integrity and PCR (amplification of a 468 bp fragment of ME.2) are shown in lanes 3, 6 and respectively 9. ME.2 and the apoCII promoter interact in antisense orientation since only the fragment of 341 bp was obtained from human monocytes and THP-1 cells differentiated with PMA (lane 2 and, respectively 5) whereas no bands were obtained for sense orientation interaction (lanes 1 and, respectively 4). In contrast, in HepG2 cells, 3C experiments gave no bands either for sense or antisense orientation interaction (lanes 7 and, respectively 8), revealing that apoCII promoter and ME.2 do not interact in hepatocytes. As control, genomic DNA isolated from THP-1 cells was used, and the amplification of the ligation products using +1378CII and +577ME.2 primers, and +1378CII and −712ME.2 primers, gave the expected size of the PCR products of 149 bp and 341 bp (Fig. 2D, lanes 10 and 11, respectively).</p