RSAD2 is abundant in atherosclerotic plaques and promotes interferon-induced CXCR3-chemokines in human smooth muscle cells.

In atherosclerotic lesions, monocyte-derived macrophages are major source of interferon gamma (IFN-γ), a pleotropic cytokine known to regulate the expression of numerous genes, including the antiviral gene RSAD2. While RSAD2 was reported to be expressed in endothelial cells of human carotid lesions, its significance for the development of atherosclerosis remains utterly unknown. Here, we harnessed publicly available human carotid atherosclerotic data to explore RSAD2 in lesions and employed siRNA-mediated gene-knockdown to investigate its function in IFN-γ-stimulated human aortic smooth muscle cells (hAoSMCs). Silencing RSAD2 in IFN-γ-stimulated hAoSMCs resulted in reduced expression and secretion of key CXCR3-chemokines, CXCL9, CXCL10, and CXCL11. Conditioned medium from RSAD2-deficient hAoSMCs exhibited diminished monocyte attraction in vitro compared to conditioned medium from control cells. Furthermore, RSAD2 transcript was elevated in carotid lesions where it was expressed by several different cell types, including endothelial cells, macrophages and smooth muscle cells. Interestingly, RSAD2 displayed significant correlations with CXCL10 (r = 0.45, p = 0.010) and CXCL11 (r = 0.53, p = 0.002) in human carotid lesions. Combining our findings, we uncover a novel role for RSAD2 in hAoSMCs, which could potentially contribute to monocyte recruitment in the context of atherosclerosis.

Hepatic stellate cell activation markers are regulated by the vagus nerve in systemic inflammation.

BACKGROUND: The liver is an important immunological organ and liver inflammation is part of the pathophysiology of non-alcoholic steatohepatitis, a condition that may promote cirrhosis, liver cancer, liver failure, and cardiovascular disease. Despite dense innervation of the liver parenchyma, little is known about neural regulation of liver function in inflammation. Here, we study vagus nerve control of the liver response to acute inflammation. METHODS: Male C57BL/6 J mice were subjected to either sham surgery, surgical vagotomy, or electrical vagus nerve stimulation followed by intraperitoneal injection of the TLR2 agonist zymosan. Animals were euthanized and tissues collected 12 h after injection. Samples were analyzed by qPCR, RNAseq, flow cytometry, or ELISA. RESULTS: Hepatic mRNA levels of pro-inflammatory mediators Ccl2, Il-1ß, and Tnf-α were significantly higher in vagotomized mice compared with mice subjected to sham surgery. Differences in liver Ccl2 levels between treatment groups were largely reflected in the plasma chemokine (C-C motif) ligand 2 (CCL2) concentration. In line with this, we observed a higher number of macrophages in the livers of vagotomized mice compared with sham as measured by flow cytometry. In mice subjected to electrical vagus nerve stimulation, hepatic mRNA levels of Ccl2, Il1ß, and Tnf-α, and plasma CCL2 levels, were significantly lower compared with sham. Interestingly, RNAseq revealed that a key activation marker for hepatic stellate cells (HSC), Pnpla3, was the most significantly differentially expressed gene between vagotomized and sham mice. Of note, several HSC-activation associated transcripts were higher in vagotomized mice, suggesting that signals in the vagus nerve contribute to HSC activation. In support of this, we observed significantly higher number of activated HSCs in vagotomized mice as compared with sham as measured by flow cytometry. CONCLUSIONS: Signals in the cervical vagus nerve controlled hepatic inflammation and markers of HSC activation in zymosan-induced peritonitis.

Cholinergic regulation of vascular endothelial function by human ChAT+ T cells.

Endothelial dysfunction and impaired vasodilation are linked with adverse cardiovascular events. T lymphocytes expressing choline acetyltransferase (ChAT), the enzyme catalyzing biosynthesis of the vasorelaxant acetylcholine (ACh), regulate vasodilation and are integral to the cholinergic antiinflammatory pathway in an inflammatory reflex in mice. Here, we found that human T cell ChAT mRNA expression was induced by T cell activation involving the PI3K signaling cascade. Mechanistically, we identified that ChAT mRNA expression was induced following the attenuation of RE-1 Silencing Transcription factor REST-mediated methylation of the ChAT promoter, and that ChAT mRNA expression levels were up-regulated by GATA3 in human T cells. In functional experiments, T cell-derived ACh increased endothelial nitric oxide-synthase activity, promoted vasorelaxation, and reduced vascular endothelial activation and promoted barrier integrity by a cholinergic mechanism. Further, we observed that survival in a cohort of patients with severe circulatory failure correlated with their relative frequency of ChAT +CD4+ T cells in blood. These findings on ChAT+ human T cells provide a mechanism for cholinergic immune regulation of vascular endothelial function in human inflammation.

Insulin Downregulates the Expression of ATP-binding Cassette Transporter A-I in Human Hepatoma Cell Line HepG2 in a FOXO1 and LXR Dependent Manner.

ATP-binding cassette transporter A-I (ABCA1) is an ubiquitously expressed protein whose main function is the transmembrane transport of cholesterol and phospholipids. Synthesis of ABCA1 protein in liver is necessary for high-density lipoprotein (HDL) formation in mammals. Thus, the mechanism of ABCA1 gene expression regulation in hepatocytes are of critical importance. Recently, we have found the insulin-dependent downregulation of other key player in the HDL formation-apolipoprotein A-I gene (J. Cell. Biochem., 2017, 118:382-396). Nothing is known about the role of insulin in the regulation of ABCA1 gene. Here we show for the first time that insulin decreases the mRNA and protein levels of ABCA1 in human hepatoma cell line HepG2. PI3K, p38, MEK1/2, JNK and mTORC1 signaling pathways are involved in the insulin-mediated downregulation of human ABCA1 gene. Transcription factors LXRα, LXRß, FOXO1 and NF-κB are important contributors to this process, while FOXA2 does not regulate ABCA1 gene expression. Insulin causes the decrease in FOXO1, LXRα and LXRß binding to ABCA1 promoter, which is likely the cause of the decrease in the gene expression. Interestingly, the murine ABCA1 gene seems to be not regulated by insulin in hepatocytes (in vitro and in vivo). We suggest that the reason for this discrepancy is the difference in the 5'-regulatory regions of human and murine ABCA1 genes.

Adiponectin Stimulates Apolipoprotein A-1 Gene Expression in HepG2 Cells via AMPK, PPARα, and LXRs Signaling Mechanisms.

Adiponectin is an adipose tissue hormone, participating in energy metabolism and involved in atherogenesis. Previously, it was found that adiponectin increases expression of the APOA1 (apolipoprotein A-1) gene in hepatocytes, but the mechanisms of this effect remained unexplored. Our aim was to investigate the role of adiponectin receptors AdipoR1/R2, AMP-activated protein kinase (AMPK), nuclear peroxisome proliferator-activated receptor alpha (PPARα) and liver X receptors (LXRs) in mediating the action of adiponectin on hepatic APOA1 expression in human hepatoma HepG2 cells. The level of APOA1 expression was determined by RT-qPCR and ELISA. We showed that the siRNA-mediated knockdown of genes coding for AdipoR1, AdipoR2, AMPK, PPARα, and LXRα and ß prevented adiponectin-induced APOA1 expression in HepG2 cells and demonstrated that interaction of PPARα and LXRs with the APOA1 gene hepatic enhancer is important for the adiponectin-dependent APOA1 transcription. The results of this study point out to the involvement of both types of adiponectin receptors, AMPK, PPARα, and LXRs in the adiponectin-dependent upregulation of the APOA1 expression.

Vagus nerve stimulation promotes resolution of inflammation by a mechanism that involves Alox15 and requires the α7nAChR subunit.

Nonresolving inflammation underlies a range of chronic inflammatory diseases, and therapeutic acceleration of resolution of inflammation may improve outcomes. Neural reflexes regulate the intensity of inflammation (for example, through signals in the vagus nerve), but whether activation of the vagus nerve promotes the resolution of inflammation in vivo has been unknown. To investigate this, mice were subjected to electrical vagus nerve stimulation (VNS) or sham surgery at the cervical level followed by zymosan-induced peritonitis. The duration of inflammation resolution was significantly reduced and efferocytosis was significantly increased in mice treated with VNS as compared with sham. Lipid mediator (LM) metabololipidomics revealed that mice treated with VNS had higher levels of specialized proresolving mediators (SPMs), particularly from the omega-3 docosahexaenoic (DHA) and docosapentaenoic (n-3 DPA) metabolomes, in peritoneal exudates. VNS also shifted the ratio between proinflammatory and proresolving LMs toward a proresolving profile, but this effect by VNS was inverted in mice deficient in 12/15-lipoxgenase (Alox15), a key enzyme in this SPM biosynthesis. The significant VNS-mediated reduction of neutrophil numbers in peritoneal exudates was absent in mice deficient in the cholinergic α7-nicotinic acetylcholine receptor subunit (α7nAChR), an essential component of the inflammatory reflex. Thus, VNS increased local levels of SPM and accelerated resolution of inflammation in zymosan-induced peritonitis by a mechanism that involves Alox15 and requires the α7nAChR.

Differentiation of human macrophages with anaphylatoxin C3a impairs alternative M2 polarization and decreases lipopolysaccharide-induced cytokine secretion.

Anaphylatoxin C3a is a small signaling polypeptide that is generated during complement activation. C3a is involved in the regulation of various innate and adaptive immune system processes; however, the role of C3a in macrophage differentiation and polarization is poorly elucidated. Here we showed that C3a impairs alternative M2 polarization of human macrophages and suppressed CD206, IL1Ra and CCL22 expression. C3a leads to a decrease of nuclear receptor PPARγ expression via the ERK1/2 signaling pathway, resulting in repressed PPARγ-dependent activation of CD36, FABP4 and LXRα genes and blunted response to an LXR ligand TO901317. Using small interfering RNA and agonist/antagonist approaches we showed that C3a decreases CD206, IL1Ra and CCL22 transcription at least partly in a PPARγ-dependent manner in M2 macrophages. Moreover, C3a impairs efferocytosis by M2 macrophages and inhibits their migratory activity. By contrast, macrophages treated with C3a during differentiation show blunted response to lipopolysaccharide stimulation owing to downregulation of TLR4 and lipid raft content. At the same time, differentiation of macrophages with C3a does not change M1 polarization in interferon gamma (IFNγ) and IFNγ + lipopolysaccharide-treated macrophages. These data provide a novel role of complement system and C3a in the regulation of M2 macrophage polarizations and suggest crosstalk between C3a, TLR4, PPARγ and LXR signaling pathways.

Regulation of Apolipoprotein A-I Gene Expression in Human Macrophages by Oxidized Low-Density Lipoprotein.

Apolipoprotein A-I (ApoA-I) is a key component of reverse cholesterol transport in humans. In the previous studies, we demonstrated expression of the apoA-I gene in human monocytes and macrophages; however, little is known on the regulation of the apoA-I expression in macrophages during the uptake of modified low-density lipoprotein (LDL), which is one of the key processes in the early stages of atherogenesis leading to formation of foam cells. Here, we demonstrate a complex nature of the apoA-I regulation in human macrophages during the uptake of oxidized LDL (oxLDL). Incubation of macrophages with oxLDL induced expression of the apoA-I gene within the first 24 hours, but suppressed it after 48 h. Both effects depended on the interaction of oxLDL with the TLR4 receptor, rather than on the oxLDL uptake by the macrophages. The oxLDL-mediated downregulation of the apoA-I gene depended on the ERK1/2 and JNK cascades, as well as on the NF-κB cascade.

AMPA-Type Glutamate Receptors Associated With Vascular Smooth Muscle Cell Subpopulations in Atherosclerosis and Vascular Injury.

Objectives and Aims: Vascular smooth muscle cells (VSMCs) are key constituents of both normal arteries and atherosclerotic plaques. They have an ability to adapt to changes in the local environment by undergoing phenotypic modulation. An improved understanding of the mechanisms that regulate VSMC phenotypic changes may provide insights that suggest new therapeutic targets in treatment of cardiovascular disease (CVD). The amino-acid glutamate has been associated with CVD risk and VSMCs metabolism in experimental models, and glutamate receptors regulate VSMC biology and promote pulmonary vascular remodeling. However, glutamate-signaling in human atherosclerosis has not been explored. Methods and Results: We identified glutamate receptors and glutamate metabolism-related enzymes in VSMCs from human atherosclerotic lesions, as determined by single cell RNA sequencing and microarray analysis. Expression of the receptor subunits glutamate receptor, ionotropic, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA)-type subunit 1 (GRIA1) and 2 (GRIA2) was restricted to cells of mesenchymal origin, primarily VSMCs, as confirmed by immunostaining. In a rat model of arterial injury and repair, changes of GRIA1 and GRIA2 mRNA level were most pronounced at time points associated with VSMC proliferation, migration, and phenotypic modulation. In vitro, human carotid artery SMCs expressed GRIA1, and selective AMPA-type receptor blocking inhibited expression of typical contractile markers and promoted pathways associated with VSMC phenotypic modulation. In our biobank of human carotid endarterectomies, low expression of AMPA-type receptor subunits was associated with higher content of inflammatory cells and a higher frequency of adverse clinical events such as stroke. Conclusion: AMPA-type glutamate receptors are expressed in VSMCs and are associated with phenotypic modulation. Patients suffering from adverse clinical events showed significantly lower mRNA level of GRIA1 and GRIA2 in their atherosclerotic lesions compared to asymptomatic patients. These results warrant further mapping of neurotransmitter signaling in the pathogenesis of human atherosclerosis.

Insulin downregulates C3 gene expression in human HepG2 cells through activation of PPARγ.

C3 is an acute phase protein, and thus its plasma concentration increases quickly and drastically during the onset of inflammation. Insulin plays a complex role in inflammation. Elevated level of plasma C3 was shown to correlate with heightened fasting insulin levels and insulin resistance and appears to be a risk factor for the cardiovascular disease and atherosclerosis. The main source of plasma C3 is liver. Nothing is known about effects of insulin on C3 gene expression and protein secretion by hepatocytes. In light of these data we asked if insulin is capable of regulating C3 production in hepatocytes. Here we show that insulin downregulates C3 gene expression in human hepatoma cells HepG2 through activation of PI3K, mTORC1, p38 and MEK1/2 signaling pathways. Transcription factors PPARα, PPARγ, HNF4α and NF-κB are important contributors to this process. Insulin activates PPARγ through PI3K/Akt/mTORC1 pathway, which results in PPARγ binding to DR4 and DR0 cis-acting elements within the C3 promoter and subsequent displacement of HNF4α and PPARα from these sites. As a result PPARα/NF-κB complex, which exists on C3 promoter, is broken down and C3 gene expression is downregulated. The data obtained can potentially be used to explain the molecular mechanism underlying the correlation between heightened level of plasma C3 and insulin resistance in humans.

Tumor necrosis factor α stimulates endogenous apolipoprotein A-I expression and secretion by human monocytes and macrophages: role of MAP-kinases, NF-κB, and nuclear receptors PPARα and LXRs.

Apolipoprotein A-I (ApoA-I) is the main structural and functional protein component of high-density lipoprotein. ApoA-I has been shown to regulate lipid metabolism and inflammation in macrophages. Recently, we found the moderate expression of endogenous apoA-I in human monocytes and macrophages and showed that pro-inflammatory cytokine tumor necrosis factor α (TNFα) increases apoA-I mRNA and stimulates ApoA-I protein secretion by human monocytes and macrophages. Here, we present data about molecular mechanisms responsible for the TNFα-mediated activation of apoA-I gene in human monocytes and macrophages. This activation depends on JNK and MEK1/2 signaling pathways in human monocytes, whereas inhibition of NFκB, JNK, or p38 blocks an increase of apoA-I gene expression in the macrophages treated with TNFα. Nuclear receptor PPARα is a ligand-dependent regulator of apoA-I gene, whereas LXRs stimulate apoA-I mRNA transcription and ApoA-I protein synthesis and secretion by macrophages. Treatment of human macrophages with PPARα or LXR synthetic ligands as well as knock-down of LXRα, and LXRß by siRNAs interfered with the TNFα-mediated activation of apoA-I gene in human monocytes and macrophages. At the same time, TNFα differently regulated the levels of PPARα, LXRα, and LXRß binding to the apoA-I gene promoter in THP-1 cells. Obtained results suggest a novel tissue-specific mechanism of the TNFα-mediated regulation of apoA-I gene in monocytes and macrophages and show that endogenous ApoA-I might be positively regulated in macrophage during inflammation.

FOXO1 and LXRα downregulate the apolipoprotein A-I gene expression during hydrogen peroxide-induced oxidative stress in HepG2 cells.

Reactive oxygen species damage various cell components including DNA, proteins, and lipids, and these impairments could be a reason for severe human diseases including atherosclerosis. Forkhead box O1 (FOXO1), an important metabolic transcription factor, upregulates antioxidant and proapoptotic genes during oxidative stress. Apolipoprotein A-I (ApoA-I) forms high density lipoprotein (HDL) particles that are responsible for cholesterol transfer from peripheral tissues to liver for removal in bile in vertebrates. The main sources for plasma ApoA-I in mammals are liver and jejunum. Hepatic apoA-I transcription depends on a multitude of metabolic transcription factors. We demonstrate that ApoA-I synthesis and secretion are decreased during H2O2-induced oxidative stress in human hepatoma cell line HepG2. Here, we first show that FOXO1 binds to site B of apoA-I hepatic enhancer and downregulates apoA-I gene activity in HepG2 cells. Moreover, FOXO1 and LXRα transcription factors participate in H2O2-triggered downregulation of apoA-I gene together with Src, JNK, p38, and AMPK kinase cascades. Mutations of sites B or C as well as the administration of siRNAs against FOXO1 or LXRα to HepG2 cells abolished the hydrogen peroxide-mediated suppression of apoA-I gene.

Insulin-Mediated Downregulation of Apolipoprotein A-I Gene in Human Hepatoma Cell Line HepG2: The Role of Interaction Between FOXO1 and LXRß Transcription Factors.

Apolipoprotein A-I (ApoA-I) is a key component of high density lipoproteins which possess anti-atherosclerotic and anti-inflammatory properties. Insulin is a crucial mediator of the glucose and lipid metabolism that has been implicated in atherosclerotic and inflammatory processes. Important mediators of insulin signaling such as Liver X Receptors (LXRs) and Forkhead Box A2 (FOXA2) are known to regulate apoA-I expression in liver. Forkhead Box O1 (FOXO1) is a well-known target of insulin signaling and a key mediator of oxidative stress response. Low doses of insulin were shown to activate apoA-I expression in human hepatoma HepG2 cells. However, the detailed mechanisms for these processes are still unknown. We studied the possible involvement of FOXO1, FOXA2, LXRα, and LXRß transcription factors in the insulin-mediated regulation of apoA-I expression. Treatment of HepG2 cells with high doses of insulin (48 h, 100 nM) suppresses apoA-I gene expression. siRNAs against FOXO1, FOXA2, LXRß, or LXRα abrogated this effect. FOXO1 forms a complex with LXRß and insulin treatment impairs FOXO1/LXRß complex binding to hepatic enhancer and triggers its nuclear export. Insulin as well as LXR ligand TO901317 enhance the interaction between FOXA2, LXRα, and hepatic enhancer. These data suggest that high doses of insulin downregulate apoA-I gene expression in HepG2 cells through redistribution of FOXO1/LXRß complex, FOXA2, and LXRα on hepatic enhancer of apoA-I gene. J. Cell. Biochem. 118: 382-396, 2017. © 2016 Wiley Periodicals, Inc.

PPARγ Represses Apolipoprotein A-I Gene but Impedes TNFα-Mediated ApoA-I Downregulation in HepG2 Cells.

Apolipoprotein A-I (ApoA-I) is the main anti-atherogenic component of human high-density lipoproteins (HDL). ApoA-I gene expression is regulated by several nuclear receptors, which are the sensors for metabolic changes during development of cardiovascular diseases. Activation of nuclear receptor PPARγ has been shown to impact lipid metabolism as well as inflammation. Here, we have shown that synthetic PPARγ agonist GW1929 decreases both ApoA-I mRNA and protein levels in HepG2 cells and the effect of GW1929 on apoA-I gene transcription depends on PPARγ. PPARγ binds to the sites A and C within the hepatic enhancer of apoA-I gene and the negative regulation of apoA-I gene transcription by PPARγ appears to be realized via the site C (-134 to -119). Ligand activation of PPARγ leads to an increase of LXRß and a decrease of PPARα binding to the apoA-I gene hepatic enhancer in HepG2 cells. GW1929 abolishes the TNFα-mediated decrease of ApoA-I mRNA expression in both HepG2 and Caco-2 cells but does not block TNFα-mediated inhibition of ApoA-I protein secretion by HepG2 cells. These data demonstrate that complex of PPARγ with GW1929 is a negative regulator involved in the control of ApoA-I expression and secretion in human hepatocyte- and enterocyte-like cells. J. Cell. Biochem. 117: 2010-2022, 2016. © 2016 Wiley Periodicals, Inc.

Hepatic nuclear factor 4α positively regulates complement C3 expression and does not interfere with TNFα-mediated stimulation of C3 expression in HepG2 cells.

Complement C3 is involved in various protective and regulatory mechanisms of immune system. Recently it was established that C3 expression is regulated by nuclear receptors. Hepatic nuclear factor 4α (HNF4α) is a nuclear receptor critical for hepatic development and metabolism. We have shown that HNF4α is a positive regulator of C3 gene expression, realizing its effects through binding to two HNF4-response elements within the C3 promoter in HepG2 cells. TNFα is a well established positive regulator of C3 expression in hepatocytes during acute phase of inflammation. TNFα decreases the amount of HNF4α protein in HepG2 cells through NF-κB and MEK1/2 pathways thereby leading to a decrease in HNF4α bound to the C3 promoter. TNFα and HNF4α act in a synergetic way resulting in the potent activation of C3 transcription. These results suggest a novel mechanism of C3 regulation during acute phase response in HepG2 cells and display the mechanism of interaction of TNFα-induced pathways and HNF4α in transcriptional regulation of C3 gene.

Peroxisome proliferator-activated receptor α positively regulates complement C3 expression but inhibits tumor necrosis factor α-mediated activation of C3 gene in mammalian hepatic-derived cells.

Complement C3 is a pivotal component of three cascades of complement activation. The liver is the main source of C3 in circulation and expression and secretion of C3 by hepatocytes is increased during acute inflammation. However, the mechanism of the regulation of the C3 gene in hepatocytes is not well elucidated. We showed that the C3 gene is the direct target for peroxisome proliferator-activated receptor α (PPARα) in human hepatoma HepG2 cells and mouse liver. Using PPARα siRNA and synthetic PPARα agonist WY-14643 and antagonist MK886 we showed that activation of PPARα results in up-regulation of C3 gene expression and protein secretion by HepG2 cells. The PPAR response element (PPRE), which is able to bind PPARα in vitro and in vivo, was found in the human C3 promoter. PPRE is conserved between human and mouse, and WY-14643 stimulates mouse C3 expression in the liver. TNFα increases C3 gene via NF-κB and, to a lesser extent, MEK1/2 signaling pathways, whereas TNFα-mediated stimulation of C3 protein secretion depends on activation of MEK1/2, p38, and JNK in HepG2 cells. Activation of PPARα abolishes TNFα-mediated up-regulation of C3 gene expression and protein secretion due to interference with NF-κB via PPRE-dependent mechanism in HepG2 cells. TNFα decreases PPARα protein content via NF-κB and MEK1/2 signaling pathways and inhibits PPARα binding with the human C3 promoter in HepG2 cells. These results suggest novel mechanism controlling C3 expression in hepatocytes during acute phase inflammation and demonstrate a crosstalk between PPARα and TNFα in the regulation of complement system.

Endogenous apolipoprotein A-I stabilizes ATP-binding cassette transporter A1 and modulates Toll-like receptor 4 signaling in human macrophages.

Apolipoprotein A-I (ApoA-I) is the main functional protein component of human high-density lipoproteins. ApoA-I shows various anti-inflammatory and atheroprotective properties toward macrophages; however, endogenous apoA-I expression has not been investigated in macrophages. We have shown that endogenous apoA-I gene is expressed in human macrophages at both mRNA and protein levels. Endogenous ApoA-I is localized in intracellular vesicles and at the external side of the plasma membrane in association with ATP-binding cassette transporter A1 (ABCA1) and lipid rafts in macrophages. We have shown that endogenous ApoA-I stabilizes ABCA1, moreover, down-regulation of ApoA-I by siRNA results in an increase of Toll-like receptor 4 (TLR4) mRNA and membrane surface protein expression, as well as an enhancement of bacterial lipopolysaccharide (LPS)-induced expression of tumor necrosis factor-α (TNF-α), interleukin 1ß (IL-1ß), and inducible nitric oxide synthase (NOS2) genes in human macrophages. TNF-α stimulates ApoA-I expression and secretion (1.2±0.2 vs. 4.3±0.9 ng/mg total protein) in macrophages. Obtained results suggest that endogenous ApoA-I has anti-inflammatory properties, presumably due to ABCA1 stabilization in macrophages; these results elucidate the cell type-specific mechanism of the TNF-α-mediated regulation of apoA-I gene expression in monocytes and macrophages.

Modified low density lipoprotein stimulates complement C3 expression and secretion via liver X receptor and Toll-like receptor 4 activation in human macrophages.

Complement C3 is a pivotal component of three cascades of complement activation. C3 is expressed in human atherosclerotic lesions and is involved in atherogenesis. However, the mechanism of C3 accumulation in atherosclerotic lesions is not well elucidated. We show that acetylated low density lipoprotein and oxidized low density lipoprotein (oxLDL) increase C3 gene expression and protein secretion by human macrophages. Modified LDL (mLDL)-mediated activation of C3 expression mainly depends on liver X receptor (LXR) and partly on Toll-like receptor 4 (TLR4), whereas C3 secretion is increased due to TLR4 activation by mLDL. LXR agonist TO901317 stimulates C3 gene expression in human monocyte-macrophage cells but not in human hepatoma (HepG2) cells. We find LXR-responsive element inside of the promoter region of the human C3 gene, which binds to LXRß in macrophages but not in HepG2 cells. We show that C3 expression and secretion is decreased in IL-4-treated (M2) and increased in IFNγ/LPS-stimulated (M1) human macrophages as compared with resting macrophages. LXR agonist TO901317 potentiates LPS-induced C3 gene expression and protein secretion in macrophages, whereas oxLDL differently modulates LPS-mediated regulation of C3 in M1 or M2 macrophages. Treatment of human macrophages with anaphylatoxin C3a results in stimulation of C3 transcription and secretion as well as increased oxLDL accumulation and augmented oxLDL-mediated up-regulation of the C3 gene. These data provide a novel mechanism of C3 gene regulation in macrophages and suggest new aspects of cross-talk between mLDL, C3, C3a, and TLR4 during development of atherosclerotic lesions.

PPARγ activates ABCA1 gene transcription but reduces the level of ABCA1 protein in HepG2 cells.

Synthesis of ABCA1 protein in liver is necessary for high-density lipoproteins (HDL) formation in mammals. Nuclear receptor PPARγ is known as activator of ABCA1 expression, but details of PPARγ-mediated regulation of ABCA1 at both transcriptional and post-transcriptional levels in hepatocytes have not still been well elucidated. In this study we have shown, that PPARγ activates ABCA1 gene transcription in human hepatoma cells HepG2 through increasing of LXRß binding with promoter region of ABCA1 gene. Treatment of HepG2 cells with PPARγ agonist GW1929 leads to dissociation of LXRß from ABCA1/LXRß complex and to nuclear translocation of this nuclear receptor resulting in reduction of ABCA1 protein level 24h after treatment. Inhibition of protein kinases MEK1/2 abolishes PPARγ-mediated dissociation of LXRß from ABCA1/LXRß complex, but does not block PPARγ-dependent down-regulation of ABCA1 protein in HepG2 cells. These data suggest that PPARγ may be important for regulation of the level of hepatic ABCA1 protein and indicate the new interplays between PPARγ, LXRß and MEK1/2 in regulation of ABCA1 mRNA and protein expression.