Possibility of increasing cholesterol efflux by adiponectin and its receptors through the ATP binding cassette transporter A1 in HEK293T cells

A decrease in adiponectin secretion leads to the early stage of atherosclerosis. Discoidal high-density lipoproteins (HDL) accept the cholesterol that effluxes from cells expressing the ATP binding cassette transporter A1 (ABCA1) in the first step of reverse cholesterol transport (RCT). Recently, a new therapeutic strategy involving reconstituted (r)HDL has been shown to enhance RCT. Therefore, we hypothesized that adiponectin may increase the efflux associated with ABCA1 and also enhance rHDL-induced efflux in human embryonic kidney 293 (HEK293T) cells. We transfected adiponectin receptor 1 and 2 (AdipoR1 and AdipoR2) cDNA into cells. The transfected cells were labeled with [³H]cholesterol following cholesterol loading with or without adiponectin for 24 h. The levels of cholesterol efflux were analyzed using a liquid scintillation counter. Treatment with adiponectin was associated with significantly higher levels of efflux in AdipoR1- and AdipoR2-transfected cells. Interestingly, rHDL-induced cholesterol efflux was enhanced in the presence of adiponectin. The down-regulation of adiponectin receptors using short-hairpin RNA decreased rHDL-induced cholesterol efflux with the down-regulation of ABCA1. In summary, adiponectin and its receptors increased cholesterol efflux and also enhanced rHDL-induced efflux at least partially through an ABCA1 pathway. These results suggest that adiponectin may enhance the RCT system and induce an anti-atherogenic effect.     

Vaccination with irradiated cercariae of Schistosoma mansoni preferentially induced the accumulation of interferon-gamma producing T cells in the skin and skin draining lymph nodes of mice

Cytokine response to schistosomula of Schistosoma mansoni was evaluated in the skin of mice during the initial 72 h following infection. These studies showed a significant increase in the levels of IL-4 and IL-10 message in the skin in areas of cercarial penetration. The IL-4 message was detectable in the skin as early as 8 h after infection and the message for IL-10 appeared from 16 h after infection. However, mRNA for IFN-gamma was undetectable in the skin samples for up to 72 h after infection with normal cercariae. In sharp contrast, vaccination with irradiated cercariae induced IFN-gamma and IL-2 responses in the skin within 24 h. Analysis of the cytokine profile of cells isolated from the skin during these early time points showed that T cells are probably not a source of IL-4 or IL-10 in the skin of mice infected with normal cercariae. However, in vaccinated animals, the majority of the IFN-gamma is derived from skin-residing T cells. In vaccinated animals, responses in the skin were mirrored in the skin-draining lymph nodes as well. Analysis of the CD4/CD8 ratio showed a significant decrease in the skin following vaccination suggesting an increase in CD8+ cells. Interestingly however, when vaccinated animals were challenged with normal cercariae, there was a significant reduction in IFN-gamma response in the skin and its draining lymph nodes. These results show that vaccination with irradiated cercariae of S. mansoni, preferentially induce the accumulation of IFN-gamma producing T cells in the skin and skin-draining lymph nodes of mice.     

Interferon-gamma down-regulates membrane adenylate cyclase activity of a murine macrophage-like cell line (P388D1)

Effects of recombinant murine interferon-gamma (rIFN-gamma) on the membrane adenylate cyclase of a murine macrophage cell line (P388D1) were investigated in order to explore the nature of a signal transmitted by IFN-gamma receptor. Following the incubation of P388D1 cells with 40 U/ml of rIFN-gamma, the intracellular level of cAMP gradually increased about twofold over the control level within 60 min, and then began to gradually decline to about half the control level by 24 h incubation. The initial rise in cAMP level appeared to be due to the modest activation of adenylate cyclase and not due to the inhibition of cAMP-phosphodiesterase. Later decrease of intracellular cAMP may be due to quantitative down-regulation of the adenylate cyclase system. The basal enzymatic activity of the membrane prepared from P388D1 cells exposed to IFN-gamma for 24 h was found to be reduced to about 20% of that of the control membrane. However, the quality of the adenylate cyclase system appeared unchanged, because the relative degree of the response of the down-regulated membrane adenylate cyclase to prostaglandin PGE2, NaF, guanylimidodiphosphate (GppNHp), choleara toxin (CT), or forskolin was found to remain unchanged. This quantitative down-regulation of adenylate cyclase must be due to the action of rIFN-gamma, since the prior treatment of rIFN-gamma with either acid (pH 2) or monoclonal anti-IFN-gamma antibody inhibited the ability of IFN-gamma to induce the down-regulation. The rIFN-gamma-induced down-regulation is a reversible process, since the adenylate cyclase activity of the membrane was found to be restored when the rIFN-gamma-exposed cells were cultured for 72 h in the absence of rIFN-gamma. In addition, the 48 h-incubation of P388D1 cells with rIFN-beta or IFN-alpha was found not to significantly affect the membrane adenylate cyclase system.     

Expression and functional analyses of novel mutations of ATP-binding cassette transporter-1 in Japanese patients with high-density lipoprotein deficiency.

ATP-binding cassette transporter-1 (ABCA1) gene is mutated in patients with familial high-density lipoprotein deficiency (FHD). In order to know the molecular basis for FHD, we characterized three different ABCA1 mutations associated with FHD (G1158A/A255T, C5946T/R1851X, and A5226G/N1611D) with respect to their expression in the passaged fibroblasts from the patients and in the cells transfected with the mutated cDNAs. Fibroblasts from the all patients showed markedly decreased cholesterol efflux to apolipoprotein (apo)-Al. In the fibroblasts homozygous for G1158A/A255T, the immunoreactive mass of ABCA1 could not be detected, even when stimulated by 9-cis-retinoic acid and 22-R-hydroxycholesterol. In the fibroblasts homozygous for C5946T/R1851X, ABCA1 mRNA was comparable. Because the mutant ABCA1 protein (R1851X) was predicted to lack the epitope for the antibody used, we transfected FLAG-tagged truncated mutant (R1851X/ABCA1-FLAG) cDNA into Cos-7 cells, showing that the mutant protein expression was markedly reduced. The expression of N1611D ABCA1 protein was comparable in both fibroblasts and overexpressing cells, although cholesterol efflux from the cells was markedly reduced. These data indicated that, in the three patients investigated, the abnormalities and dysfunction of ABCA1 occurred at the different levels, providing important information about the expression, regulation, and function of ABCA1.     

Anti-CD3 antibody induces rapid expression of cytokine genes in vivo

Anti-CD3 antibody was administered to mice i.v. and the kinetics of spleen cell cytokine mRNA expression studied by Northern analysis. Untreated mice and mice receiving control antibody had low or undetectable amounts of mRNA for c-fos, c-myc, IL-2, IL-4, and IFN-gamma. After injection of anti-CD3 antibody, substantial increases in all were found. Induction of c-fos was detected at 10 min and of c-myc at 30 min after injection. IL-2, IL-4, and IFN-gamma mRNA were induced by 30 min and reached peak levels at 60 min. Thereafter, IL-2 and IL-4 mRNA declined, whereas IFN-gamma mRNA persisted. The induced cytokine mRNA was not observed in athymic nu/nu mice nor in normal spleen cells from which T cells had been depleted in vitro. The early in vivo induction of IL-4 mRNA contrasts with prior in vitro studies in which IL-4 production was difficult to detect after primary stimulation. To assess the possibility that many T cells had been preprimed in vivo, germfree mice were compared with conventional mice and no differences in cytokine mRNA were found. These data show that T cell-dependent IL message production can be induced rapidly in vivo without prepriming and that the cytokine messages induced after anti-CD3 antibody administration do not suggest a predominance of either Th1 or Th2 type cells.     

A biochemical signature for rapid recall of memory CD4 T cells

Mechanisms for the rapid recall response mediated by memory T cells remain unknown. In this study, we present a novel, multiparameter analysis of TCR-coupled signaling and function in resting and activated naive and memory CD4 T cells, revealing a biochemical basis for immunological recall. We identify a striking elevation in expression of the proximal tyrosine kinase Zap70 in resting Ag-specific and polyclonal mouse memory vs naive CD4 T cells that is stably maintained independent of protein synthesis. Elevated Zap70 protein levels control effector function as IFN-gamma production occurs exclusively from the Zap70(high) fraction of activated T cells in vitro and in vivo, and specific down-modulation of Zap70 expression in memory CD4 T cells by small interfering RNA or protein inhibition significantly reduces rapid IFN-gamma production. Downstream of Zap70, we show quantitative differences in distal phosphorylation associated with effector function in naive and memory subsets, with low accumulation of phosphorylation in memory T cells producing IFN-gamma at early time points, contrasting extensive phosphorylation associated with IFN-gamma production following sustained activation of naive T cells. Our results reveal a novel biochemical signature imparted to memory CD4 T cells enabling efficacious responses through increased Zap70 expression and reduced accumulation of downstream signaling events.     

Regulation of cholesterol distribution in macrophage-derived foam cells by interferon-gamma

The Th1-derived cytokine gamma interferon, IFN-gamma, is present within the microenvironment of an atheromatous lesion and likely contributes to lesion progression through macrophage activation. While the inflammatory effects of IFN-gamma are well known, the role of this cytokine in cholesterol metabolism in macrophage derived foam cells is unclear. In the present study, the incubation of foam cells with IFN-gamma resulted in the reduction of HDL(3)-mediated cholesterol efflux. The decrease in cholesterol efflux was not observed with other macrophage-activating factors as colony-stimulating factors failed to demonstrate a similar effect. The reduction in cholesterol efflux was independent of apoE synthesis or SR-BI expression and was associated with a redistribution of intracellular cholesterol with an increase in cholesteryl ester accumulation. The increase in the esterified pool, primarily in cholesterol eicosapentadenoate, docosapentaenoate, arachidonate, and linoleate was associated with a 2-fold increase in acyl-CoA:cholesterol-O-acyltransferase, ACAT, activity and message without any change in neutral cholesteryl ester hydrolase activity. While CD36 message was reduced in IFN-gamma-treated foam cells, the ability to reverse the decrease in efflux by the ACAT inhibitor A58035 in a dose-dependent manner suggests that the IFN-gamma effect on efflux is primarily through the modulation of ACAT expression. Therefore, in addition to its inflammatory effects, IFN-gamma can contribute to the progression of an atherosclerotic lesion by altering the pathway of intracellular cholesterol trafficking in macrophage derived foam cells.     

Interferon-gamma reduces cell surface expression of annexin 2 and suppresses the invasive capacity of prostate cancer cells

The effect of interferon-gamma (IFNgamma) treatment on cell surface protein expression was studied in the human prostate cancer cell line 1542CP3TX. IFNgamma increased both the number and abundance of proteins in membrane fractions. In contrast, the expression of annexin 2 and its binding partner p11 decreased by 4-fold after 24 h of exposure, with the remaining anx2(t) complexes localized to lipid rafts. Within the same time scale, IFNgamma reduced the abundance of the peripherally attached, anx2(t)-associated proteases procathepsin B and plasminogen. The invasive capacity of the cancer cells was reduced by treatment with IFNgamma or antibody to annexin 2 in 1542CP3TX cells, but not in LNCaP, an annexin 2-negative prostate cancer cell line. Expression of annexin 2 in LNCaP cells increased their invasiveness. IFNgamma induced calpain expression and activation and increased the phosphorylation and degradation of the calpain substrate ABCA1 in 1542CP3TX cancer cells. Surface expression of annexin 2 was reduced in cells treated with glyburide, an ABCA1 inhibitor, whereas inhibition of calpain abrogated IFNgamma-induced annexin 2 down-regulation and suppression of Matrigel invasion. The findings suggest annexin 2 externalization is coupled to lipid efflux in prostate epithelium and that IFNgamma induces down-regulation of the protease-binding anx2(t) scaffold at the cell surface and consequently acts to suppress invasiveness through calpain-mediated degradation of the lipid transporter ABCA1.     

Activation of liver X receptor decreases BACE1 expression and activity by reducing membrane cholesterol levels

The synthetic Liver X receptor (LXR) activator T0901317 has been reported to exert neuroprotective effect in Alzheimer’s disease, but the relationship between LXR activation and beta-site amyloid precursor protein cleaving enzyme 1 (BACE-1) remains uncertain. This study investigated the effect of T0901317 on membrane cholesterol levels, BACE1 expression and activity. We found that T0901317 decreased membrane cholesterol levels, reduced BACE1 expression and activity as well as β-secretase cleaved C-terminal fragment (β-CTF) levels in vivo and in vitro. Meanwhile, the expression of ATP-binding membrane cassette transport protein A1 (ABCA1) enhanced. Additionally, inhibition of ABCA1 abrogated the effects of T0901317 on membrane cholesterol levels and β-secretase activity. Moreover, addition of LXR antagonist reversed the effect of T0901317 on ABCA1 mRNA expression, membrane cholesterol levels and β-secretase activity. Our results suggest that activation of LXR may decrease BACE1 expression and activity through a pathway associated with ABCA1-mediated reduction in membrane cholesterol levels.     

A role for inducible costimulator protein in the CD28- independent mechanism of resistance to Toxoplasma gondii

Long-term resistance to Toxoplasma gondii is dependent on the development of parasite-specific T cells that produce IFN-gamma. CD28 is a costimulatory molecule important for optimal activation of T cells, but CD28(-/-) mice are resistant to T. gondii, demonstrating that CD28-independent mechanisms regulate T cell responses during toxoplasmosis. The identification of the B7-related protein 1/inducible costimulator protein (ICOS) pathway and its ability to regulate the production of IFN-gamma suggested that this pathway may be involved in the CD28-independent activation of T cells required for resistance to T. gondii. In support of this hypothesis, infection of wild-type or CD28(-/-) mice with T. gondii resulted in the increased expression of ICOS by activated CD4(+) and CD8(+) T cells. In addition, both costimulatory pathways contributed to the in vitro production of IFN-gamma by parasite-specific T cells and when both pathways were blocked, there was an additive effect that resulted in almost complete inhibition of IFN-gamma production. Although in vivo blockade of the ICOS costimulatory pathway did not result in the early mortality of wild-type mice infected with T. gondii, it did lead to increased susceptibility of CD28(-/-) mice to T. gondi associated with reduced serum levels of IFN-gamma, increased parasite burden, and increased mortality compared with the control group. Together, these results identify a critical role for ICOS in the protective Th1-type response required for resistance to T. gondii and suggest that ICOS and CD28 are parallel costimulatory pathways, either of which is sufficient to mediate resistance to this intracellular pathogen.     

Activation of protein kinase C down-regulates IFN-gamma receptors

Treatment of mouse EL-4 cells with intracellular activators of protein kinase C, namely 4-phorbol 12-myristate 13-acetate (PMA) and diacylglycerol, resulted in 90% reduction in cell surface interferon-gamma (IFN-gamma) receptors as judged by iodinated-IFN-gamma binding. This did not seem to be due to a decreased in the receptor affinity, since that of the remaining surface receptors appeared to be significantly increased as shown in Scatchard plot analysis. Kinetics experiments revealed that a PMA treatment as short as 15 min was sufficient to induce a decrease of 30% of IFN-gamma receptors, whereas the highest levels of down-regulation were observed after 60-90 min. Treatment of EL-4 cells with calcium ionophore, A23187, although ineffective by itself, dramatically increased the ability of suboptimal PMA concentrations to mediate IFN-gamma receptor down-regulation. Finally, specificity studies revealed that PMA is particularly effective in decreasing the binding of IFN-gamma to T-lymphocytes. Altogether these results suggest a possible involvement of protein kinase C in the regulation of IFN-gamma receptor expression.     

Phorbol Ester- and Retinoic Acid-Induced Regulation of the Protein Kinase C Substrate MARCKS in Immortalized Hippocampal Cells

Abstract: The expression of MARCKS, a major protein kinase C (PKC) substrate, was examined in the immortalized hippocampal cell line HN33, following differentiation using phorbol esters or retinoic acid. In cells exposed to phorbol esters, MARCKS protein levels were reduced through an apparent PKC-dependent mechanism. Exposure to 1 µ M phorbol 12-myristate 13-acetate (PMA) for 10 min resulted in a rapid loss of PKC activity in the soluble fraction with a concurrent increase in membrane-associated PKC activity. PKC activity was reduced to <20% of control values in both soluble and membrane fractions following 1 h of PMA exposure. Significant reductions in MARCKS protein levels were initially observed in membrane and soluble fractions following PMA exposure for 4 and 8 h, respectively. The reduction in MARCKS protein levels was maximal following 24 h of PMA exposure. MARCKS protein expression was also down-regulated in a dose-dependent manner on exposure of HN33 cells to retinoic acid. In cells exposed to 10 µ M retinoic acid, the MARCKS protein level was reduced in the membrane fraction within 4 h. Reduction of MARCKS protein levels was maximal (>90%) by 12 h with no evidence for any alteration in PKC activity. Reduced levels of MARCKS protein were also observed in the soluble fraction of retinoic acid-exposed cells, but to a significantly lesser extent. Addition of the PKC inhibitor GF109203X blocked the down-regulation of MARCKS protein in PMA-treated cultures but not in retinoic acid-treated cells. These findings suggest that the down-regulation of MARCKS may play an important role in both phorbol ester- and retinoic acid-induced differentiation in cells of neuronal origin.     

Interleukin-10 inhibits the down-regulation of ATP binding cassette transporter A1 by tumour necrosis factor-alpha in THP-1 macrophage-derived foam cells

It is suggested that cholesterol efflux mediated by ATP binding cassette transporter A1 (ABCA1) plays an important role in anti-atherogenesis. However, the effects of inflammatory cytokines on ABCA1 expression and cholesterol accumulation in foam cells are little known. This study investigates the effects of tumour necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) on ABCA1 expression and cholesterol content in THP-1 macrophage-derived foam cells. ABCA1mRNA and protein levels were determined by RT-PCR and Western blot, respectively. The total cholesterol content in THP-1 macrophage-derived foam cells was detected by the zymochemistry method. Results revealed that TNF-alpha could increase cholesterol content by down-regulating ABCA1 expression in a time-dependent manner in THP-1 macrophage-derived foam cells, which may contribute to its pro-atherosclerotic effect. In addition IL-10 time-dependently decreased cholesterol accumulation by up-regulating ABCA1 expression and inhibited the down-regulation of ABCA1 by TNF-alpha in THP-1 macrophage-derived foam cells, which may be one of the mechanisms of IL-10 contributing to its anti-atherosclerotic action.     

Type 2 diabetes is associated with reduced ATP-binding cassette transporter A1 gene expression, protein and function

Objective

Increasing plasma glucose levels are associated with increasing risk of vascular disease. We tested the hypothesis that there is a glycaemia-mediated impairment of reverse cholesterol transport (RCT). We studied the influence of plasma glucose on expression and function of a key mediator in RCT, the ATP binding cassette transporter-A1 (ABCA1) and expression of its regulators, liver X receptor-α (LXRα) and peroxisome proliferator-activated receptor–γ (PPARγ).

Methods and Results

Leukocyte ABCA1, LXRα and PPARγ expression was measured by polymerase chain reaction in 63 men with varying degrees of glucose homeostasis. ABCA1 protein concentrations were measured in leukocytes. In a sub-group of 25 men, ABCA1 function was quantified as apolipoprotein-A1-mediated cholesterol efflux from 2–3 week cultured skin fibroblasts. Leukocyte ABCA1 expression correlated negatively with circulating HbA1c and glucose (rho = −0.41, p<0.001; rho = −0.34, p = 0.006 respectively) and was reduced in Type 2 diabetes (T2DM) (p = 0.03). Leukocyte ABCA1 protein was lower in T2DM (p = 0.03) and positively associated with plasma HDL cholesterol (HDL-C) (rho = 0.34, p = 0.02). Apolipoprotein-A1-mediated cholesterol efflux correlated negatively with fasting glucose (rho = −0.50, p = 0.01) and positively with HDL-C (rho = 0.41, p = 0.02). It was reduced in T2DM compared with controls (p = 0.04). These relationships were independent of LXRα and PPARγ expression.

Conclusions

ABCA1 expression and protein concentrations in leukocytes, as well as function in cultured skin fibroblasts, are reduced in T2DM. ABCA1 protein concentration and function are associated with HDL-C levels. These findings indicate a glycaemia- related, persistent disruption of a key component of RCT.