MCP-1 Promoter Research Articles

Effects of Inflammatory Factor Expression Regulated by 12/15 Lipoxygenase on Obesity-Related Nephropathy.

Background: It has been demonstrated that 12/15-lipoxygenase (LO) contributes to insulin resistance by promoting beta cells’ exposure to inflammation. We investigate the mechanism by which 12/15-LO regulates the expression of inflammatory factors in obesity-related glomerular disease (ORG). Methods: Glomerular mesangial cells were treated with metabolite of 12/15-LO, and the expression of inflammatory factors was measured. Cell histones methylation in 12/15-LO related metabolic memory process were evaluated by chromatin immunoprecipitation (ChIP) assays. Wild-type (WT) and 12/15-LO knockout mice were fed a high-fat diet (HFD) to induce ORG. Results: 12(S)-HETE increased TNF-α, MCP-1, and IL-6 mRNA expression. Inhibition of 12/15-LO reduced the expression of inflammatory factors stimulated by PA or TNF-α. ChIP assays showed that 12(S)-HETE increased H3K4me modification in the TNF-α, IL-6, and MCP-1 gene promoters, and decreased H3K9me3 modification in the MCP-1 and IL-6 gene promoter. Urinary albumin excretion was greater in HFD-fed than in standard fat diet-fed mice, but both urinary protein and microalbumin amounts were lower in HFD-fed 12/15-LO knockout than in WT mice. The levels of TNF-α, IL-6, and MCP-1 in serum and renal cortex were higher in WT than in 12/15-LO knockout mice. Conclusions: 12/15-LO may regulate the expression of inflammatory factors in ORG by methylation of histones in the promoter regions of genes encoding inflammatory factors, sustaining the inflammatory phenotype of ORG.

Lack of association between methylation status of CpG in the MCP-1 promoter and type 2 diabetes mellitus and its complications in a Moroccan population

BackgroundMonocyte chemoattractant protein −1 (MCP-1) plays an important role in the development of diseases related to metabolic syndromes, such as type 2 diabetes mellitus (T2DM) and its complications. The present study investigates the relationship between the methylation status of CpG sites in the MCP-1 promoter and T2DM and its complications in Morocco. Material and methodsWe conducted a population-based case-control study of 108 T2DM cases and 101 controls. Methylation Specific Polymerase chain reaction (MSP) was used to analyze the MCP-1 methylation profile in leucocyte's DNA. ResultsThis study did not indicate a significant association between the methylation status of CpG sites in the MCP1 promoter and T2DM and its complications (p ˃ 0.05). The hypomethylation profile is significantly associated with higher LDL-C levels (142.37 ± 58.37; p = 0.041) and treatment with antidepressants (18.8%; p = 0.043) in patients with T2DM. The hypermethylation profile is most frequently observed in type 2 diabetic patients with complications and cigarettes smokers (31.6%; p = 0.052). ConclusionResults of our study showed no association between the methylation status of CpG in the MCP-1 promoter and T2DM and its complications in the Moroccan population. However, the methylation status might be related to LDL-C levels, treatment with antidepressants, and smoking status.

KLF6 Acetylation Promotes Sublytic C5b-9-Induced Production of MCP-1 and RANTES in Experimental Mesangial Proliferative Glomerulonephritis

Rat Thy-1 nephritis (Thy-1N) is an experimental mesangial proliferative glomerulonephritis (MsPGN) for studying human MsPGN. Although sublytic C5b-9 complex formation on glomerular mesangial cells (GMCs) and renal MCP-1 and RANTES production in rats with Thy-1N have been proved, the role and mechanism of MCP-1 or RANTES synthesis in GMCs induced by sublytic C5b-9 are poorly elucidated. In this study, we first found the expression of transcription factor (KLF6), co-activator (KAT7) and chemokines (MCP-1 and RANTES) was all up-regulated both in renal tissue of Thy-1N rats (in vivo) and in sublytic C5b-9-induced GMCs (in vitro). Further in vitro experiments revealed that KLF6 bound to MCP-1 promoter (-297 to -123 nt) and RANTES promoter (-343 to -191 nt), leading to MCP-1 and RANTES gene transcription. Meanwhile, KAT7 also bound to the same region of MCP-1 and RANTES promoter in a KLF6-dependent manner, and KLF6 was acetylated by KAT7 at lysine residue 100, which finally promoted MCP-1 and RANTES expression. Moreover, our in vivo experiments discovered that knockdown of renal KAT7 or KLF6 gene obviously reduced MCP-1 and RANTES production, GMCs proliferation, ECM accumulation, and proteinuria secretion in Thy-1N rats. Collectively, our study indicates that sublytic C5b-9-induced MCP-1 and RANTES synthesis is associated with KAT7-mediated KLF6 acetylation and elevated KLF6 transcriptional activity, which might provide a new insight into the pathogenesis of rat Thy-1N and human MsPGN.

Esculetin ameliorates vascular perturbation by intervening in the occupancy of H2BK120Ub at At1, At2, Tgfβ1 and Mcp1 promoter gene in thoracic aorta of IR and T2D rats

Micro and macro vascular complications under diabetic condition are the responses to pathological stimuli exerted by up regulated renin angiotensin system (RAS) via deteriorating vascular physiology. Up-regulated RAS could influence in the adaptive mechanisms of target tissues to alter the abundance of angiotensin II type 1 receptor (AT1) and angiotensin II type 2 receptor (AT2). Such differential regulation of AT1 and AT2 have been reported to be associated with post-translational histone modifications (PTHMs). Additionally, recent evidences provide, esculetin (6,7-dihydroxycoumarin) reverses post-translational histone modifications (PTHMs) in diabetic cardiomyopathy and nephropathy. On account of these evidences, we further pursued this study to investigate the effects of esculetin on PTHMs in progressive vascular complications under insulin resistance (IR) and type 2 diabetic (T2D) conditions. Esculetin treatment in both IR and T2D conditions substantially improved vascular reactivity, increased eNos and decreased Vcam1 mRNA levels, and reduced collagen deposition in rat thoracic aorta. Further, the fold changes in At1 and At2 receptor mRNA in IR and T2D were reversed by esculetin treatment. Modifications in histone H2B lysine 120 monoubiquitination (H2BK120Ub) were also reversed in esculetin treatment group. Further, modification in the occupancy of H2BK120Ub at At1a, At2, Tgfβ and Mcp1 promoter genes was evidenced by treatment with esculetin. Taken together, our investigation concluded with the involvement of esculetin in the amelioration of vascular perturbation by modifying H2BK120Ub along with occupancy at At1, At2, Tgfβ1 and Mcp1 promoter gene.

Abstract 5924: Necrotic cells promote microglia infiltration in glioblastoma through regulating MCP-1 and MIP-3α expression

Abstract Glioblastoma multiform (GBM), a grade IV astrocytoma, is most lethal and common adult primary intracranial tumor. GBM is characterized by diffuse infiltration into normal brain parenchyma, rapid growth and the presence of necrosis and microglia/macrophage infiltration. Among these properties of GBM, necrosis has been implicated to be a strong predictor of poor prognosis; however, the effect of necrosis on GBM progression is poorly understood at present. In this study, we examined the effect of necrosis on glioblastoma cells by exploring molecular mechanisms underlying gene expression and chemokine expression. Data obtaining from chemokine array and ELISA showed that CRT-MG human gliomablastoma cells secreted several chemokines including MCP-1 and MIP-3α in response to necrotic cells. Expression levels of mRNA and protein of MCP-1 and MIP-3α were also increased by treatment with necrotic cells in CRT-MG in a dose-dependent manner. Necrotic cells induced NF-κB/AP-1 activation and their binding to the MCP-1 and MIP-3α promoter, leading to enhanced MCP-1 and MIP-3α production in GBM cells. Finally, transwell migration assay showed that incubation with necrotic cells significantly enhanced the migration of microglia. Our data demonstrate that when GBM cells are exposed to and stimulated by necrotic cells, the expression and secretion of MCP-1 and MIP-3α are enhanced in GBM cells and microglia infiltration/migration to the tumor site are facilitated. Citation Format: Yieun Jung, So-Hee Ahn, Hyunju Park, Jiwoo Lim, Jihee Lee Kang, Youn-Hee Choi, Eun Ju Kim. Necrotic cells promote microglia infiltration in glioblastoma through regulating MCP-1 and MIP-3α expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5924. doi:10.1158/1538-7445.AM2017-5924

Spatially and Temporally Regulated NRF2 Gene Therapy Using Mcp-1 Promoter in Retinal Ganglion Cell Injury

Retinal ganglion cell degeneration triggered by axonal injury is believed to underlie many ocular diseases, including glaucoma and optic neuritis. In these diseases, retinal ganglion cells are affected unevenly, both spatially and temporally, such that healthy and unhealthy cells coexist in different patterns at different time points. Herein, we describe a temporally and spatially regulated adeno-associated virus gene therapy aiming to reduce undesired off-target effects on healthy retinal neurons. The Mcp-1 promoter previously shown to be activated in stressed retinal ganglion cells following murine optic nerve injury was combined with the neuroprotective intracellular transcription factor Nrf2. In this model, Mcp-1 promoter-driven NRF2 expression targeting only stressed retinal ganglion cells showed efficacy equivalent to non-selective cytomegalovirus promoter-driven therapy for preventing cell death. However, cytomegalovirus promoter-mediated NRF2 transcription induced cellular stress responses and death of Brn3A-positive uninjured retinal ganglion cells. Such undesired effects were reduced substantially by adopting the Mcp-1 promoter. Combining a stress-responsive promoter and intracellular therapeutic gene is a versatile approach for specifically targeting cells at risk of degeneration. This strategy may be applicable to numerous chronic ocular and non-ocular conditions.

The -2518 A/G MCP-1 and -403 G/A RANTES promoter gene polymorphisms are associated with psoriasis vulgaris.

Polymorphic variants of the genes encoding monocyte chemoattractant protein-1 (MCP-1/CCL2) and regulated upon activation normal T-cell expressed and secreted (RANTES/CCL5) and their protein serum levels have not been widely explored in psoriasis. To clarify the effect of the MCP-1 (-2518 A/G) and RANTES (-403 G/A) promoter gene polymorphisms on the risk and clinical manifestation of psoriasis. We enrolled 160 unrelated patients with psoriasis vulgaris and 160 healthy, unrelated, age- and sex-matched volunteers. The promoter gene polymorphisms were analysed using amplification refractory mutation system (ARMS)-PCR and single specific primer (SSP)-PCR. Serum levels of cytokines were measured using ELISA. The presence of the MCP-1-2518 GG genotype was statistically more frequent in patients and it was associated with an increased risk of psoriasis (OR = 1.94; P = 0.04). In patients with late-onset (≥ 40 years) psoriasis, the presence of the RANTES -403 AA genotype was statistically more frequent (OR = 3.65; P < 0.01) while -403 GG was less frequent (OR = 0.44; P < 0.01). Moreover, the A allele (AA or AG) in the -403 RANTES polymorphism was associated with an increased risk of developing severe psoriasis (OR = 2.02; P = 0.03). Serum levels of both chemokines were elevated. RANTES serum concentration was significantly higher in patients with Psoriasis Area and Severity Index > 15. The results of our analysis suggest that the -2518 A/G MCP-1 and -403 G/A RANTES promoter gene polymorphisms may be risk factors for psoriasis and may influence its clinical presentation.

H2AK119 monoubiquitination regulates Angiotensin II receptor mediated macrophage infiltration and renal fibrosis in type 2 diabetic rats.

Monocyte chemoattractant protein (MCP-1) and transforming growth factor-β (TGF-β1)-markers of inflammation and fibrosis, are central to type 2 diabetic nephropathy (T2DN) progression. The epigenetic basis of their expression has also been explored to certain extent. H2A lysine 119 monoubiquitination (H2AK119Ub), a repressive chromatin mark regulates progression of hyperglycaemia induced fibrosis in glomerular mesangial cells. However, how H2AK119Ub affects the expression of MCP-1 and TGF-β1 and their regulation by Angiotensin II receptor subtypes remains unknown. In the current study, we aimed to study the effect of Angiotensin II receptors' blockade on the macrophage infiltration and histone modifications occurring at the promoter region of Mcp1 and Tgfb1in high fat diet fed and low dose streptozotocin treated male Wistar rats. Hereby, we present the first report delineating a distinct link between H2AK119Ub and macrophage infiltration and fibrosis i.e. the enrichment of H2AUb at Mcp1 and Tgfb1 promoter region was found to reduce drastically in the T2DN which could be significantly reversed by Telmisartan and was further elevated by PD123319. We could conclude that the Angiotensin II mediated macrophage infiltration in T2DN is regulated at least partially by H2AK119Ub through both AT1 and AT2 receptors, which to the best of our knowledge, presents the first report for the regulation of Mcp1 by H2AK119Ub. Thus an approach targeting AT1R blockade and AT2R activation accompanied by an epigenetic modulator may be more suitable to ameliorate the macrophage infiltration and fibrosis associated with T2DN.

A peptide tetramer Tk-tPN induces tolerance of cardiac allografting by conversion of type 1 to type 2 immune responses via the Toll-like receptor 2 signal-promoted activation of the MCP1 gene.

The plant protein trichosanthin (Tk) and its derived peptide tetramer Tk-tPN have been shown to stimulate the type 2 immune responses for treating autoimmune disease. This work explores the possibility of using Tk-tPN as a non-toxic immunosuppressant to induce transplantation tolerance using the mechanisms by which T-cell-mediated immune responses are transferred from type 1 to type 2 through innate immunity-related pathways. Immunocytes and cytokine secretions involved in the mouse cardiac allografting model with Tk-tPN treatment were characterized. Identification of critical genes and analysis of their functions through Toll-like receptor (TLR) -initiated signalling and the possible epigenetic changes were performed. Mean survival times of the cardiac allografts were delayed from 7.7 ± 0.3 days (control) to 22.7 ± 3.9 days (P < 0.01) or 79.1 ± 19.2 days (P < 0.0001) when Tk-tPN was introduced into the recipients alone or together with rapamycin, respectively. The grafting tolerance was donor-specific. The secretion pattern of the type 1 cytokine/transcription factor (IL-2(+) IFN-γ(+) T-bet(+)), which is responsible for the acute graft rejection, was shifted to the type 2 factor (IL-4(+) IL-10(+) Gata3+), together with a selective expansion of the IL-4/IL-10-producing CD8+ CD28- regulatory T-cell subset. A TLR2-initiated high expression of chemokine gene MCP1 was detectable simultaneously. Epigenetically Tk/Tk-tPN could also acetylate the histone H3K9 of MCP1 promoter to skew the immunity towards T helper type 2 responses. Tk/Tk-tPN is therefore capable of down-regulating the type 1 response-dominant rejection of cardiac allografts by evoking type 2 immunity through the activation of a TLR2-initiated signalling pathway and MCP1 gene to expand the IL-4/IL-10-secreting CD8+ CD28- regulatory T cells. Tk-tPN could be a promising novel immunosuppressant to induce tolerance in allotransplantation.

Endothelial cell tumor growth is Ape/ref-1 dependent.

Tumor-forming endothelial cells have highly elevated levels of Nox-4 that release H2O2 into the nucleus, which is generally not compatible with cell survival. We sought to identify compensatory mechanisms that enable tumor-forming endothelial cells to survive and proliferate under these conditions. Ape-1/ref-1 (Apex-1) is a multifunctional protein that promotes DNA binding of redox-sensitive transcription factors, such as AP-1, and repairs oxidative DNA damage. A validated mouse endothelial cell (EOMA) tumor model was used to demonstrate that Nox-4-derived H2O2 causes DNA oxidation that induces Apex-1 expression. Apex-1 functions as a chaperone to keep transcription factors in a reduced state. In EOMA cells Apex-1 enables AP-1 binding to the monocyte chemoattractant protein-1 (mcp-1) promoter and expression of that protein is required for endothelial cell tumor formation. Intraperitoneal injection of the small molecule inhibitor E3330, which specifically targets Apex-1 redox-sensitive functions, resulted in a 50% decrease in tumor volume compared with mice injected with vehicle control (n = 6 per group), indicating that endothelial cell tumor proliferation is dependent on Apex-1 expression. These are the first reported results to establish Nox-4 induction of Apex-1 as a mechanism promoting endothelial cell tumor formation.

L-cystathionine inhibits oxidized low density lipoprotein-induced THP-1-derived macrophage inflammatory cytokine monocyte chemoattractant protein-1 generation via the NF-κB pathway.

This study aimed to explore whether and how L-cystathionine had any regulatory effect on the inflammatory response in THP-1-derived macrophages cultured in vitro under oxidized low-density lipoprotein (ox-LDL) stimulation. The human monocyte line THP-1 cell was cultured in vitro and differentiated into macrophages after 24 hours of PMA induction. Macrophages were pretreated with L-cystathionine and then treated with ox-LDL. The results showed that compared with the controls, ox-LDL stimulation significantly upregulated the expression of THP-1-derived macrophage MCP-1 by enhancing NF-κB p65 phosphorylation, nuclear translocation and DNA binding with the MCP-1 promoter. Compared with the ox-LDL group, 0.3 mmol/L and 1.0 mmol/L L-cystathionine significantly inhibited the expression of THP-1-derived macrophage MCP-1. Mechanistically, 0.3 mmol/L and 1.0 mmol/L L-cystathionine suppressed phosphorylation and nuclear translocation of the NF-κB p65 protein, as well as the DNA binding activity and DNA binding level of NF-κB with the MCP-1 promoter, which resulted in a reduced THP-1-derived macrophage MCP-1 generation. This study suggests that L-cystathionine could inhibit the expression of MCP-1 in THP-1-derived macrophages induced by ox-LDL via inhibition of NF-κB p65 phosphorylation, nuclear translocation, and binding of the MCP-1 promoter sequence after entry into the nucleus.

Fli-1 controls transcription from the MCP-1 gene promoter, which may provide a novel mechanism for chemokine and cytokine activation

Regulation of proinflammatory cytokines and chemokines is a primary role of the innate immune response. MCP-1 is a chemokine that recruits immune cells to sites of inflammation. Expression of MCP-1 is reduced in primary kidney endothelial cells from mice with a heterozygous knockout of the Fli-1 transcription factor. Fli-1 is a member of the Ets family of transcription factors, which are evolutionarily conserved across several organisms including Drosophilla, Xenopus, mouse and human. Ets family members bind DNA through a consensus sequence GGAA/T, or Ets binding site (EBS). Fli-1 binds to EBSs within the endogenous MCP-1 promoter by ChIP assay. In this study, transient transfection assays indicate that the Fli-1 gene actively promotes transcription from the MCP-1 gene promoter in a dose-dependent manner. Mutation of the DNA binding domain of Fli-1 demonstrated that Fli-1 activates transcription of MCP-1 both directly, by binding to the promoter, and indirectly, likely through interactions with other transcription factors.Another Ets transcription factor, Ets-1, was also tested, but failed to promote transcription. While Ets-1 failed to drive transcription independently, a weak synergistic activation of the MCP-1 promoter was observed between Ets-1 and Fli-1. In addition, Fli-1 and the NFκB family member p65 were found to interact synergistically to activate transcription from the MCP-1 promoter, while Sp1 and p50 inhibit this interaction. Deletion studies identified that EBSs in the distal and proximal MCP-1 promoter are critical for Fli-1 activation from the MCP-1 promoter. Together, these results demonstrate that Fli-1 is a novel regulator of the proinflammatory chemokine MCP-1, that interacts with other transcription factors to form a complex transcriptional mechanism for the activation of MCP-1 and mediation of the inflammatory response.

Thicker Carotid Intima Media Thickness in Children with Monocyte Chemoattractant Protein-1: A-2138T and A-2464G Mutation.

Carotid intima media thickness (CIMT) is clearly associated with atherosclerosis. Studies in ischemic stroke patients reveal that there is a significant association between CIMT with monocyte chemoattractant protein-1 (MCP-1) and osteopontin (OPN) promoter polymorphism. This research aims to explain the effect of MCP-1 and OPN promoter polymorphism toward CIMT changes identified in Javanese Indonesian children. Subjects were 54 children: 27 were from parents with ischemic stroke (cases), and 27 were from healthy parents (controlled). The CIMT was examined by utilizing high resolution B-mode ultrasound. Physical examination and genotyping analysis of MCP-1 promoter were conducted by employing PCR method. Research results indicate that two polymorphisms were obtained, that is, A-2138T and G-2464A, respectively. A-2138T polymorphism was found in 5% of case children and in 14.3% of controlled children. G-2464A polymorphism was found in 5% of case children. CIMT of case children was significantly different from that of controlled children (0.61 ± 0.012 mm versus, 0.52 ± 0.015 mm, P = 0.021). Subjects with MCP-1 promoter polymorphism have 1.471 times higher tendency to have thicker CIMT than subjects with no polymorphism in MCP1 promoter. OPN promoter T-66G was also studied but it did not indicate occurrence of polymorphism in samples.

Fli-1 drives transcription from the MCP-1 gene promoter, which may provide a novel mechanism for cytokine and chemokine activation. (P6236)

Abstract Regulation of proinflammatory cytokines and chemokines is a primary role of the innate immune response. MCP-1 is a chemokine that recruits immune cells to sites of inflammation. Expression of MCP-1 is reduced in primary kidney endothelial cells from mice with a heterozygous knockout of the Fli-1 transcription factor. Fli-1 is a member of the Ets family of transcription factors, which are evolutionarily conserved across several organisms including Drosophilla, Xenopus, mouse and human. Ets family members bind DNA through a consensus sequence GGAA/T, or Ets binding site (EBS). We have demonstrated that Fli-1 binds to three EBSs within the endogenous MCP-1 promoter by ChIP assay. Transient transfection assays of the murine MCP-1 promoter indicate that the Fli-1 gene actively drives transcription from the MCP-1 gene promoter in a dose-dependent manner. The Ets-1 transcription factor was also tested, but failed to drive transcription. Studies to determine the relationship between Fli-1 and Ets-1 in the transcriptional control of MCP-1 will be presented. Deletion studies to determine the most important regions of the MCP-1 promoter for Fli-1 gene regulation are being investigated. Together, these results demonstrate a novel pathway, separate from NFκB, for the transcriptional activation of MCP-1. Evidence suggests that Fli-1 may interact with the promoters of other chemokines and cytokines, providing a novel mechanism for activation of the inflammatory response.

IFNβ autocrine feedback is required to sustain TLR induced production of MCP-1 in macrophages

Chemokines, including MCP-1, are crucial to mounting an effective immune response due to their ability to recruit other immune cells. We show that sustained LPS or poly(I:C)-stimulated MCP-1 production requires an IFNβ-mediated feedback loop. Consistent with this, exogenous IFNβ was able to induce MCP-1 transcription in the absence of other stimuli. Blocking IFNβ signaling with Ruxolitinib, a JAK inhibitor, inhibited MCP-1 transcription. The MCP-1 promoter contains potential STAT binding sites and we demonstrate that STAT1 is recruited upon IFNβ stimulation. Furthermore we find that IL-10 knockout increases MCP-1 production in response to LPS, which may reflect an ability of IL-10 to repress IFNβ production. Overall, these results show the importance of the balance between IFNβ and IL-10 in the regulation of MCP-1.

Bindarit

The activation of nuclear factor (NF)κB pathway and its transducing signaling cascade has been associated with the pathogenesis of many inflammatory diseases. The central role that IκBα and p65 phosphorylation play in regulating NFκB signalling in response to inflammatory stimuli made these proteins attractive targets for therapeutic strategies. Although several chemical classes of NFκB inhibitors have been identified, it is only for a few of those that a safety assessment based on a comprehensive understanding of their pharmacologic mechanism of action has been reported. Here, we describe the specific anti-inflammatory effect of bindarit, an indazolic derivative that has been proven to have anti-inflammatory activity in a variety of models of inflammatory diseases (including lupus nephritis, arthritis and pancreatitis). The therapeutic effects of bindarit have been associated with its ability to selectively interfere with monocyte recruitment and the "early inflammatory response," although its specific molecular mechanisms have remained ill-defined. For this purpose, we investigated the effect of bindarit on the LPS-induced production of inflammatory cytokines (MCP-1 and MCPs, IL-12β/p40, IL-6 and IL-8/KC) in both a mouse leukaemic monocyte-macrophage cell line and bone marrow derived macrophages (BMDM). Bindarit inhibits the LPS-induced MCP-1 and IL-12β/p40 expression without affecting other analyzed cytokines. The effect of bindarit is mediated by the downregulation of the classical NFκB pathway, involving a reduction of IκBα and p65 phosphorylation, a reduced activation of NFκB dimers and a subsequently reduced nuclear translocation and DNA binding. Bindarit showed a specific inhibitory effect on the p65 and p65/p50 induced MCP-1 promoter activation, with no effect on other tested activated promoters. We conclude that bindarit acts on a specific subpopulation of NFκB isoforms and selects its targets wihtin the whole NFκB inflammatory pathway. These findings pave the way for future applications of bindarit as modulator of the inflammatory response.

Overexpression of Receptor for Advanced Glycation End Products Induces Monocyte Chemoattractant Protein-1 Expression in Rat Vascular Smooth Muscle Cell Line

The receptor for advanced glycation end-products (RAGE) has been suggested to play a pivotal role in the development of diabetic vasculopathy and atherosclerosis; however, due to its low expression, the physiological role of RAGE in vascular smooth muscle cells (VSMC) remains unknown. Using VSMC lines stably expressing RAGE (RAGE-A10), we studied the molecular mechanism by which S100B, a RAGE ligand, induces proinflammatory gene expression. S100B induced NF-κB activation and the expression of several proinflammatory genes (MCP-1, IL-6, ICAM-1) at mRNA and protein levels in RAGE-A10, among which MCP-1 expression was the most robust. S100B-induced MCP-1 expression was dose-dependently blocked by inhibitors of JNK (SP600125), p38 (SB203580), MEK-1 (U0126) as well as NF-κB (Bay117085). In RAGE-A10, S100B activated JNK, MEK-1 and p38. S100B-induced MCP-1 promoter activity via NF-κB binding sites and nuclear translocation of NF-κB p65 subunit were blocked by SP600125, U0126, and SB203580 in RAGE-A10. Our study demonstrates that S100B increased MCP-1 expression via NF-κB and mitogen-activated protein kinase (JNK, ERK1/2, and p38) pathways in RAGE-overexpressed A10 cell lines. Thus, RAGE-A10 could be a useful cell model for studying the molecular mechanism(s) of up-regulated RAGE in the vasculature.

Postprandial activation of protein kinase C� regulates the expression of adipocytokines via the transcription factor AP-2β

Abnormal secretion of adipocytokines promotes atherosclerosis, diabetes and insulin resistance, and is mainly induced by adipocyte hypertrophy. Recently, the circulating adipocytokine concentrations were reported to change in the postprandial period, as the levels of TNFα, IL-6 IL-8 and MCP-1 increased after a meal, whereas that of adiponectin decreased. These data suggest that prandial modulation of cytokines may be involved in the pathogenesis of atherosclerosis in type 2 diabetes. However, the regulatory mechanism of such change is still unclear. In the present study, we identified this mechanism with a special focus on the functions of protein kinase C (PKC) and of the transcription factor AP-2β, both of which are associated with the pathophysiology of adipocytokine regulation. PKCµ was highly phosphorylated in the re-feeding condition compared to the fasting condition in mouse adipose tissue, while other PKC isoforms remained unchanged. Furthermore, overexpression of PKCµ in 3T3-L1 adipocytes, but not other PKC isoforms, positively regulated the mRNA expression and promoter activity of MCP-1 and IL-6, and negatively regulated those of adiponectin. AP-2β had similar effects on the expression and promoter activity of these adipocytokines. Interestingly, overexpression of PKCµ enhanced the stimulatory and inhibitory effects of AP-2β on the expression of these adipocytokines. Finally, PKCµ could not activate a mutant MCP-1 promoter lacking the AP-2β binding domain. Our results suggest that postprandial activation of PKCµ plays a role in disordered postprandial adipocytokine expression through AP-2β.

Effect of CC Chemokine Receptor 2 CCR2 Blockade on Serum C-Reactive Protein in Individuals at Atherosclerotic Risk and With a Single Nucleotide Polymorphism of the Monocyte Chemoattractant Protein-1 Promoter Region

CC chemokine receptor 2 (CCR2), expressed on the surface of circulating monocytes, and its ligand monocyte chemoattractant protein-1 (MCP-1; also known as CC-chemokine ligand 2) are present in atherosclerotic plaques and may have important roles in endothelial monocyte recruitment and activation. MLN1202 is a highly specific humanized monoclonal antibody that interacts with CCR2 and inhibits MCP-1 binding. The aim of this randomized, double-blind, placebo-controlled study was to measure reductions in circulating levels of high-sensitivity C-reactive protein, an established biomarker of inflammation associated with coronary artery disease, on MLN1202 treatment in patients at risk for atherosclerotic cardiovascular disease (≥2 risk factors for atherosclerotic cardiovascular disease and circulating high-sensitivity C-reactive protein >3 mg/L). Additionally, patients were genotyped for the 2518 A→G polymorphism in the promoter of the MCP-1 gene to investigate the correlation between this polymorphism and reduced C-reactive protein levels with MLN1202 treatment. Patients who received MLN1202 exhibited significant decreases in high-sensitivity C-reactive protein levels, beginning at 4 weeks and continuing through 12 weeks after dosing. Patients with A/G or G/G genotypes in the MCP-1 promoter had significantly greater reductions in high-sensitivity C-reactive protein levels than patients with the wild-type A/A genotype. In conclusion, MLN1202 treatment was well tolerated in this patient population and resulted in significant reductions in high-sensitivity C-reactive protein levels.

RNA-destabilizing Factor Tristetraprolin Negatively Regulates NF-κB Signaling

Tristetraprolin (TTP) is a CCCH zinc finger-containing protein that destabilizes mRNA by binding to an AU-rich element. Mice deficient in TTP develop a severe inflammatory syndrome mainly because of overproduction of tumor necrosis factor alpha. We report here that TTP also negatively regulates NF-kappaB signaling at the transcriptional corepressor level, by which it may repress inflammatory gene transcription. TTP expression inhibited NF-kappaB-dependent transcription. However, overexpression of TTP did not affect reporter mRNA stability. Instead, TTP functioned as a corepressor of p65/NF-kappaB. In support of this concept, we found that TTP physically interacted with the p65 subunit of NF-kappaB and was also associated with HDAC1, -3, and -7 in vivo. Treatment with histone deacetylase inhibitors or small interfering RNA induced HDAC1 or HDAC3 knockdown completely or partly abolished the inhibitory activity of TTP on NF-kappaB reporter activation. Consistently, chromatin immunoprecipitation showed decreased recruitment of HDAC1 and increased recruitment of CREB-binding protein on the Mcp-1 promoter in TTP(-/-) cells compared with wild-type cells. Moreover, overexpression of TTP blocked CREB-binding protein-induced acetylation of p65/NF-kappaB. Taken together, these data suggest that TTP may also function in vivo as a modulator in suppressing the transcriptional activity of NF-kappaB.