Yonghae Son

Roles of MAPK and NF-κB in Interleukin-6 Induction by Lipopolysaccharide in Vascular Smooth Muscle Cells

Toll-like receptor (TLR)-4 signaling promotes cytokine synthesis in vascular smooth muscle cells (VSMC). However, it is unknown how TLR-4 regulates interleukin-6 (IL-6) in VSMC. Therefore, the present study investigated cellular factors involved in TLR-4-mediated IL-6 in VSMC in terms of MAPK and transcription elements. Exposure of aortic smooth muscle cells to TLR4-specific lipopolysaccharide (LPS) not only enhanced IL-6 release but also induced IL-6 transcript via promoter activation. The promoter activation was attenuated by dominant-negative MKK1 and to a lesser extent by dominant-negative MKK3, but not by dominant-negative MKK4. IL-6 promoter activity was diminished by U0126 or SB202190, but not by SP600125. Co-transfection with dominant negative CCAAT/enhancer binding protein or with IκB suppressed LPS-induced promoter activation, whereas the promoter activity was not influenced by dominant negative c-Jun. Mutation in the IL-6 promoter region at the binding site of NF-κB or C/EBP impaired promoter activation in response to LPS. Further impairment occurred when both NF-κB- and C/EBP-binding sites were mutated. LPS-induced IL-6 promoter activation was also prevented by pretreatment with epigallocatechin 3-gallate, curcumin, and resveratrol. The present study reports that TLR4-agonistic LPS induces IL-6 through transcriptional activation in VSMC and ERK1/2, p38 MAPK, NF-κB, and C/EBP play active roles in that process.

7-Ketocholesterol Upregulates Interleukin-6 via Mechanisms That Are Distinct from Those of Tumor Necrosis Factor-α, in Vascular Smooth Muscle Cells

This study investigated the effects of 7-ketocholesterol on interleukin (IL)-6 expression in vascular smooth muscle cells (VSMC). Among the 7 IL examined, only IL-6 transcript was increased by 7-ketocholesterol treatment in human aorta smooth muscle cells. IL-6 transcripts increased up to 24 h after treatment with 7-ketocholesterol, and this effect was profoundly repressed by treatment with p38 MAPK inhibitors and to a lesser extent JNK inhibitors. 7α-Hydroxycholesterol, 27-hydroxycholesterol or cholesterol, however, did not induce IL-6 expression. Mechanisms of IL-6 induction by 7-ketocholesterol were investigated in comparison with tumor necrosis factor (TNF)-α. Whereas TNF-α activated IL-6 promoter, which was impaired by p38 MAPK inhibitors or by mutation in the NF-κB-binding site within the promoter region, 7-ketocholesterol did not affect IL-6 promoter activity. Instead, this oxysterol slowed degradation of IL-6 mRNA and increased the amount of cytoplasmic HuR. 7-ketocholesterol significantly increased the amount of intracellular IL-6 protein in the presence of brefeldin A. 7-Ketocholesterol also enhanced IL-6 release from VSMC. IL-6 release by 7-ketocholesterol, although significant, was not as remarkable as that induced by TNF-α. These data suggest that 7-ketocholesterol upregulates IL-6 via mechanisms distinct from TNF-α and contributes to the intra- and extracellular IL-6 deposits within the vasculature.

27-hydroxycholesterol induces production of tumor necrosis factor-alpha from macrophages.

Enhanced production of TNF-α from macrophages promotes development and instability of atherosclerotic plaques, but involvement of lipid component in TNF-α production has not been clarified in atherosclerosis. We attempted to determine whether cholesterol oxidation products (oxysterols) could modify TNF-α production. Treatment of THP-1 cells with 27-hydroxycholesterol (27OHChol) or 7α-hydroxycholesterol (7αOHChol) resulted in a profound increase in TNF-α transcription, while treatment with an identical concentration of cholesterol and 7-ketochoelsterol did not lead to any change in TNF-α expression. Treatment with 27OHChol resulted in increased synthesis, as well as secretion, of TNF-α, while 7αOHChol led to increased synthesis of TNF-α without affecting secretion of the cytokine. Co-treatment with 7αOHChol or 27OHChol and LPS resulted in synergistically enhanced or augmented secretion of TNF-α. Treatment with TO-901317, pertussis toxin, PP2, and LY294002 resulted not only in attenuated transcription of TNF-α induced by 27OHChol and 7αOHChol, but also secretion of TNF-α enhanced by 27OHChol. This is the first report demonstrating enhanced production of TNF-α in macrophages by treatment with oxysterols which are detected in abundance in atheromatous lesions; in addition, results of the current study provide evidence indicating that certain types of oxysterols contribute to development of atherosclerosis by promoting production of proinflammatory cytokines.

Peptidoglycan enhances secretion of monocyte chemoattractants via multiple signaling pathways.

Peptidoglycan (PG) is detected in a high proportion in inflammatory cell-rich regions of human atheromatous plaques. In the present study, we determined the cellular factors involved in PG-mediated chemokine expression in mononuclear cells in order to understand the molecular mechanisms of inflammatory responses to bacterial pathogen-associated molecular patterns in the diseased artery. Exposure of human monocytic leukemia THP-1 cells to PG resulted in not only enhanced secretion of CCL2 and CCL4 but also profound induction of their gene transcripts, which were abrogated by oxidized 1-palmitoyl-2-arachidonosyl-sn-phosphatidylcholine, an inhibitor of Toll-like receptors (TLRs)-2/4, but not by polymyxin B. PG enhanced phosphorylation of Akt and mitogen-activated protein kinases and activated protein kinase C. Pharmacological inhibitors such as SB202190, SP6001250, U0126, Akt inhibitor IV, rapamycin, and RO318220 significantly attenuated PG-mediated up-regulation of CCL2 and CCL4. We propose that PG contributes to vascular inflammation in atherosclerotic plaques by upregulating expression of mononuclear cell chemoattractants via TLR-2, protein kinase C, Akt, mTOR, and mitogen-activated protein kinases.

Oxysterols induce transition of monocytic cells to phenotypically mature dendritic cell-like cells

Dendritic cells (DCs) activate adaptive immune responses in atherosclerotic plaques; however, the origin of DCs is in question. We attempted to determine whether cholesterol or its oxide forms, which are detected in abundance in atheromatous lesions, could induce differentiation or transition of monocytic cells to DCs. Treatment of THP-1 cells with 27-hydroxycholesterol (27OH-Chol) and 7α-hydroxycholesterol (7αOH-Chol) resulted in an increase in the numbers of adherent cells, and, in contrast to PMA, decreased uptake of FITC-conjugated dextran. In addition, treatment with 27OH-Chol and 7αOH-Chol induced expression of mDC-specific molecules, including CD40, CD80, CD83, and CD88. Of the two oxysterols, 27OH-Chol enhanced expression of MHC class I and II molecules as well as CCR7. However, treatment with an identical concentration of cholesterol and 7-ketocholesterol did not influence adherence, uptake of FITC-conjugated dextran, and expression of the aforementioned molecules. This is the first study to report on change of monocytic cells by oxysterols to phenotypically atypical cells with some characteristics of mDCs detected in atherosclerotic lesions. We propose that a certain type of oxysterol would contribute to immune responses in atherosclerotic lesions by enhancing expression of multiple CD molecules as well as MHC molecules by monocytic cells.

Cellular factors involved in CXCL8 expression induced by glycated serum albumin in vascular smooth muscle cells

Glycated serum albumin (GSA) promotes vascular complications in diabetes. The aim of this study was to determine if GSA induces chemokine, particularly CXCL8 (IL-8), and to determine intracellular signaling pathways activated by GSA in vascular smooth muscle cells (VSMCs). GSA increased IL-8 transcription via promoter activation and enhanced CXCL8 release from VSMCs. GSA-induced promoter activation of the IL-8 gene was suppressed by dominant-negative mutants of TLR-4, MyD88, and TRIF, but not by a dominant-negative form of TLR-2. In addition, IL-8 up-regulation in response to GSA was inhibited by resveratrol, curcumin, diphenyleneiodium, U0126, and SB202190. Mutation at the NF-kappaB- or C/EBP-binding site, but not at the AP-1-binding site, in the IL-8 promoter region suppressed GSA-induced promoter activation. Moreover, gene delivery of IkappaB suppressed CXCL8 release. This study suggests that GSA induces expression of IL-8 in VSMCs and that TLR-4, mitogen-activated protein kinases, NF-kappaB, and NADPH oxidase are involved in that process.

ATF3 plays a role in adipocyte hypoxia-mediated mitochondria dysfunction in obesity.

Obesity-associated adipose tissue hypoxia plays a pivotal role in insulin resistance via impaired adipocyte dysfunction including mitochondria dysfunction. In this study, we investigated the involvement of hypoxia-inducible ATF3 in adipocyte hypoxia-mediated mitochondrial dysfunction. While HIF-1α and ATF3 were increased in white adipose tissue of high fat diet (HFD) obese mice compared with control lean mice, mitochondria-related genes were significantly reduced. Treatment with hypoxia mimetics CoCl(2) or incubation with 2% O(2) impaired mitochondria function as demonstrated by decreases in ATP production, NADH dehydrogenase activity, mitochondrial membrane potential, and reduced expression of mitochondria-related genes including NRF-1, PGC-1α, COX1 and SOD in 3T3-L1 adipocyte cells. Furthermore, overexpression of ATF3 in 3T3-L1 cells also decreased mitochondria function as well as expression of mitochondria-related genes. ATF3 knockdown in 3T3-L1 cells partly prevented the hypoxia-mediated decrease in mitochondria function and expression of mitochondria-related genes. The mitochondria-related genes were decreased in white adipose tissue of ATF3-overexpressing mice compared with wild-type mice. These results suggest that ATF3 may play a role in adipocyte hypoxia-mediated mitochondrial dysfunction in obesity.

7α-Hydroxycholesterol induces inflammation by enhancing production of chemokine (C-C motif) ligand 2.

We investigated pro-inflammatory activity of 7-oxygenated cholesterol derivatives present in atherosclerotic lesions. Treatment of THP-1 monocyte/macrophage with 7α-hydroxycholesterol (7αOHChol) resulted in increased gene transcription of CCL2 and production of its corresponding protein. The conditioned medium isolated from THP-1 cells treated with 7αOHChol enhanced migration of monocytic cells, and migration was inhibited in the presence of CCL2-neutralizing antibody. In contrast, 7β-hydroxycholesterol (7βOHChol) or 7-ketocholesterol (7K) did not induce expression of CCL2, and the conditioned medium isolated from THP-1 cells exposed to 7βOHChol or 7K did not affect migration of monocytic cells. 7αOHChol also enhanced production of MMP-9. Inhibition of MEK or PI3K resulted in significantly attenuated expression of CCL2, along with that of MMP-9, induced by 7αOHChol. We propose that elevated concentration of a certain type of 7-oxygenated cholesterol derivative, like 7αOHChol, leads to inflammation via upregulation of CCL2 and MMP-9 in macrophages in the artery, thereby promoting progression of atherosclerosis, and the ERK and the PI3K pathways are involved in the process.

7-Ketocholesterol induces the reduction of KCNMB1 in atherosclerotic blood vessels.

Hypertension is a high-risk symptom in atherosclerotic patients, and vascular rigidity is one of the main factors leading to hypertension. β1-Subunit of BKCa channel (KCNMB1; MaxiKβ1) has been reported as a modulator of vascular flexibility. To determine the relationship between atherosclerosis and KCNMB1, we studied some atherogenic factors affecting vascular tone. Blood of atherosclerotic patients shows increased concentration of 7-ketocholesterol (7K), which has been studied as a harmful lipid to blood vessels. Our data showed that KCNMB1 was significantly down-regulated in the presence of 7K, in a dose-/time-dependent manner in vascular smooth muscle cells (VSMCs). And, the reduction of KCNMB1 was confirmed in cell images of 7K-stimulated VSMCs and in vessel tissue images of ApoE knock-out mice. To determine whether aryl hydrocarbon receptor (AhR) was involved in the reduction of KCNMB1 by 7K-stimulation, protein level of AhR was analyzed by Western blot. Our data showed that the reduction of KCNMB1 was modulated through the AhR pathway. In conclusion, results of our study suggest that 7K induces the reduction of KCNMB1 through the AhR pathway.

Roles of 7-ketocholesterol on the homeostasis of intracellular cholesterol level.

Abstract: Atherosclerotic plaque contains materials, such as cholesterol, oxysterols, cell debris, modified fatty acids, and infiltrated cells. Among them, cholesterol is the major component in plaque. Cholesterol is known to originate from the influx of extracellular materials, but this explanation is not enough for the cholesterol accumulation observed in atherosclerotic plaque. This study examined the origins of cholesterols in plaques. The main focus was to determine if the intracellular cholesterol levels are affected by oxysterols in human vascular smooth muscle cells. The results showed that the cholesterol levels increased in response to a 7-ketocholesterol (7K)-treatment in a dose-dependent manner. Eight enzymes involved in cholesterol biosynthesis were examined. Among them, squalene epoxidase (SQLE) was increased by 7K but not by 7&agr;-hydroxycholesterol, 27-hydroxycholesterol (27OH-chol), or cholesterol. The 7K-induced SQLE expression was suppressed in the presence of the enzyme inhibitor SB203580 but not by UO126 and SP600125. The SQLE immunoreactivity was detected in the atherosclerotic plaque of the aortic roots from apoE−/− mice. In addition, 7K increased the cholesterol level and SQLE expression in murine bone marrow–derived macrophages. This suggests that 7K increases the intracellular cholesterol level through an elevation of SQLE expression, which might affect the progress of cholesterol accumulation in the atherosclerotic lipid core.