Interleukin-6 Gene Transcription Research Articles

Modulation of IL-6 Expression by KLF4-Mediated Transactivation and PCAF-Mediated Acetylation in Sublytic C5b-9-Induced Rat Glomerular Mesangial Cells.

Interleukin-6 (IL-6) overproduction has been considered to contribute to inflammatory damage of glomerular mesangial cells (GMCs) in human mesangial proliferative glomerulonephritis (MsPGN) and its rat model called Thy-1 nephritis (Thy-1N). However, the regulatory mechanisms of IL-6 expression in GMCs upon sublytic C5b-9 timulation remain poorly understood. We found that Krüppel-like factor 4 (KLF4) bound to the IL-6 promoter (−618 to −126 nt) and activated IL-6 gene transcription. Furthermore, lysine residue 224 of KLF4 was acetylated by p300/CBP-associated factor (PCAF), which was important for KLF4-mediated transactivation. Moreover, lysine residue 5 on histone H2B and lysine residue 9 on histone H3 at the IL-6 promoter were also acetylated by PCAF, which resulted in an increase in IL-6 transcription. Besides, NF-κB activation promoted IL-6 expression by elevating the expression of PCAF. Overall, these findings suggest that sublytic C5b-9-induced the expression of IL-6 involves KLF4-mediated transactivation, PCAF-mediated acetylation of KLF4 and histones, and NF-κB activation in GMCs.

Respiratory virus induced IL‐6 expression is regulated by an extragenic suppressor

Interleukin‐6 (IL‐6) is a pleiotropic inflammatory cytokine that plays a pivotal role in the activation and regulation of proper immune response during respiratory viral infections. Aberrant IL‐6 expression, however, may promote virus survival and/or exacerbation of clinical symptoms, such as the inflammatory cytokine storms observed in severe COVID‐19 patients infected with SARS‐CoV‐2. IL‐6 is now regarded as a prominent target for clinical intervention but less is known about how IL‐6 itself is regulated in various disorders.Using precision nuclear run‐on next generation sequencing (PRO‐Seq) to measure the transcriptionally engaged RNA polymerases (RNA Pol II) in human primary small airway epithelial cells (hSAECs), we have discovered an upstream enhancer‐like region in the IL‐6 gene locus. This is a region that resides within an open chromatin domain. It has DNase I hypersensitivity and enriches in activating H3K27Ac mark, but lack of active transcription mark H3K36me3. Interestingly, it displays increased ratio of H3K4me1 over H3K4me3, a phenomenon usually associated with active enhancers. We were therefore surprised to observe that rhinovirus (RV) infection disrupts active RNA Pol II binding in this region, despite the fact that RNA Pol II is increased in the body of the IL‐6 gene. These findings suggest that the upstream sequence may be functioning as a suppressor of IL‐6 expression. To test this possibility, we targeted the KRAB transcriptional silencer to the upstream sequence using CRISPRi (KRAB/dCas9 directed by sgRNA). hSAECs expressing control sgRNA or the sequence‐specific sgRNA were infected with RV and IL‐6 mRNA measured by Q‐RT‐PCR. Intriguingly, the sequence‐specific sgRNA enhanced RV‐ induced IL‐6 expression. These results suggest that the upstream element is not an enhancer but a functionally active suppressor of IL‐6 expression and that the mechanism of IL‐6 expression involves a previously unknown de‐repression step. It will advance us to understand how dynamic interactions between cis‐regulatory elements regulate the threshold, magnitude and duration of IL‐6 gene transcription in response to distinct factors.

Regulation of stimulus-induced interleukin-8 gene transcription in human adrenocortical carcinoma cells – Role of AP-1 and NF-κB

Stimulation of H295R adrenocortical carcinoma cells with angiotensin II or cytokines induces the secretion of the chemokine interleukin-8 (IL-8). Here, we have analyzed the molecular mechanism of stimulus-induced IL-8 expression. IL-8 expression and IL-8 promoter activity increased in H295R cells expressing an activated Gαq-coupled designer receptor. H295R cells stimulated with either interleukin-1β (IL-1β) or phorbol ester also showed elevated IL-8 mRNA levels and higher IL-8 promoter activities. Deletion and point mutations of the IL-8 promoter revealed that the AP-1 binding site within the IL-8 promoter is essential to connect designer receptor stimulation with the transcriptional activation of the IL-8 gene. Expression of a constitutively active mutant of c-Jun, or expression of constitutively active mutants of the protein kinases MEKK1 and MKK6 confirmed that the IL-8 gene is a bona fide target of AP-1 in adrenocortical carcinoma cells. Upregulation of IL-8 expression in IL-1β-treated H295R cells required NF-κB while the phorbol ester TPA used both the AP-1 and NF-κB sites of the IL-8 gene to stimulate IL-8 expression. These data were corroborated in experiments with chromatin-embedded AP-1 or NF-κB-responsive reporter genes. While stimulation of Gαq-coupled designer receptors increased the AP-1 activity in the cells, IL-1β specifically stimulated NF-κB-regulated transcription. Stimulation of the cells with TPA increased both AP-1 and NF-κB activities. We conclude that stimulation of Gαq-coupled designer receptors or IL-1 receptors triggers distinct signaling pathways in H295R cells leading to the activation of either AP-1 or NF-κB. Nevertheless, both signaling cascades converge to the IL-8 gene, inducing IL-8 gene transcription.

The Effect of Habitual Fat Intake, IL6 Polymorphism, and Different Diet Strategies on Inflammation in Postmenopausal Women with Central Obesity.

The hypothesis that habitual fat intake, the IL6 genotype, the Mediterranean diet or the central European diet for 16 weeks affect biomarkers of inflammation in centrally obese postmenopausal women, was tested in a randomized controlled trial. Dietary intake was assessed using a three-day food diary. Lipid parameters were measured using a Beckman Coulter AU analyzer. Transcription of TNF and IL6 genes was analyzed in peripheral blood mononuclear cells using real-time PCR. Concentrations of tumor necrosis factor alpha (TNFα) and interleukin 6 (IL6) were measured with ELISA. rs1800795 polymorphism of IL6 was analyzed using hydrolyzing probes. Higher energy intake from fat was associated with higher IL6 levels (p < 0.05). Significantly (p < 0.01) lower total cholesterol (T-C) and low-density lipoprotein cholesterol (LDL-C) concentrations were observed in the GG IL6 rs1800795 genotype group. Both diets significantly (p < 0.001) decreased TNFα concentrations. Neither IL6 gene transcription levels nor blood IL6 concentrations were affected by them. Our findings confirm that habitual fat intake may affect inflammation. The rs1800795 IL6 polymorphism alone did not significantly affect body weight or body composition in aimed group, but C-allele carriers had higher levels of T-C and LDL-C. This polymorphism did not affect inflammation. Both diets may lead to a decrease in TNFα concentration.

Analysis of Cell Viability and Gene Expression After Continuous Ultrasound Therapy in L929 Fibroblast Cells.

This study aimed to analyze cell viability and gene expression interleukin 6 and vascular endothelial growth factor after continuous ultrasound therapy of 1 and 3 MHz in L929 fibroblast cells. The L929 cells were cultivated in 12-well plates and divided into the following five groups: Group 1 (G1), nonirradiated; G2, 0.2 W/cm-1 MHz; G3, 0.5 W/cm-1 MHz; G4, 0.2 W/cm-3 MHz; and G5, 0.5 W/cm-3 MHz. The cells were irradiated at 24 and 48 hrs. Cell viability was evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method. The gene expression analysis was assessed using real-time polymerase chain reaction. The G2 and G3 showed a decrease in cell viability when compared with the G1 at 48 hrs (P < 0.01). The G4 and G5 presented an increase in viability (P = 0.01 and P = 0.03, respectively) in 24 to 48 hrs. The cells irradiated at an intensity of 0.5 W/cm-3 MHz at 48 hrs showed a 0.21-fold decrease in interleukin-6 gene transcripts and a 1.67-fold increase in vascular endothelial growth factor gene transcripts. Continuous ultrasound therapy with a frequency of 3 MHz at an intensity of 0.5 W/cm stimulates cell proliferation, decreases interleukin-6 gene expression, and increases vascular endothelial growth factor gene expression in L929 fibroblast cells.

Stimulation of transient receptor potential M3 (TRPM3) channels increases interleukin-8 gene promoter activity involving AP-1 and extracellular signal-regulated protein kinase

Stimulation of Ca2+ permeable TRPM3 (transient receptor potential melastatin-3) channels with the steroid ligand pregnenolone sulfate activates stimulus-responsive transcription factors, including the transcription factor AP-1 (activator protein-1). As part of a search for AP-1-regulated target genes we analyzed the gene encoding interleukin-8 (IL-8) in HEK293 cells expressing TRPM3 channels. Here, we show that stimulation of TRPM3 channels activated transcription of an IL-8 promoter-controlled reporter gene that was embedded into the chromatin of the cells. Mutational analysis of the IL-8 promoter revealed that the AP-1 binding site of the IL-8 promoter was essential to connect TRPM3 stimulation with the transcription of the IL-8 gene. Genetic experiments revealed that the basic region leucine zipper proteins c-Jun and ATF2 and the ternary complex factor Elk-1 are essential to couple TRPM3 channel stimulation with the IL-8 gene. Moreover, we identified extracellular signal-regulated protein kinase (ERK1/2) as signal transducer connecting TRPM3 stimulation with enhanced transcription of the IL-8 gene. Furthermore, we show that stimulation of TRPC6 (transient receptor potential canonical-6) channels with its ligand hyperforin also increased IL-8 promoter activity, involving the AP-1 binding site within the IL-8 gene, suggesting that activation of IL-8 gene transcription may be a common theme following TRP channel stimulation.

The Role of Interleukin-8 and Its Mechanism in Patients with Breast Cancer: Its Relation with Oxidative Stress and Estrogen Receptor

Context: Breast cancer is one of the leading causes of death among women all over the world. Prognostic markers can be used for patient survival. Likewise, these biomarkers are able to generate main information related to clinical manner of breast gland tumors. Interleukin-8 (IL-8) is a chemotactic cytokines caused initiation of an inflammatory response. Evidence Acquisition: We performed a computerized search in Medline/PubMed databases with key words: IL-8, breast cancer, and oxidative stress. Results: IL-8 secretion motivates the expression of adhesion molecule, including fibronectin in human inflammatory breast cancer cells. IL-8 activates PI3K/Akt pathway, which in turn activates NF-κB resulted up-regulation of integrin β3 expression and increases the invasion of breast cancer cells. Therefore, it seems that IL-8/PI3K/Akt/NF-κB/integrin β3 axis may be used for therapeutic intervention of breast cancer with metastasis status. NF-κB plays an important role in IL-8 gene transcription via several signals, such as LPS, TNF-alpha, and oxidative stress. Oxidative stress caused angiogenic factor production, such as vascular endothelial growth factor and IL-8 via cancer cells. Moreover, there is inverse relation between ER level and IL-8, but the mechanism of IL-8 action in estrogen receptor-negative breast cancer is largely unknown. Estrogen receptor and growth factor in breast cancer cells is influenced by mitogen activated protein kinase (MAPK) cascade, as a part of IL-8 signaling pathway. Conclusions: According to these studies, interleukin-8 may be as a main biomarker for breast cancer. It seems that signaling pathway of IL-8 may be used for therapeutic intervention of breast cancer. Also oxidative stress and breast cancer cells with lacking estrogen receptor generate more interleukin-8.

Influence of the IL6 − 147C/G polymorphism on clinical characteristics of chronic hepatitis C in Brazilian patients

Interleukin-6 (IL-6) is a pleotropic cytokine which plays an important role in the immunological response to infections. Single nucleotide polymorphism at the promoter (−174C/G) of the IL-6 gene was associated with IL-6 gene transcription rate and high circulating levels of IL-6 appears to affect the outcome of hepatitis C virus (HCV) infection. Therefore, the aim of our study was to ascertain if the IL-6 polymorphism is associated with HCV infection and clinical characteristics in Brazilian patients. Patients with chronic hepatitis C, infected with HCV genotypes 2 or 3, were studied. The IL-6 −174C/G polymorphism (rs1800795) was analyzed by polymerase chain reaction (PCR) followed by restriction digestion. Viral genotype was obtained from medical records. A total of 125 patients and 125 healthy blood donor controls were evaluated. HCV genotype 2 was found in 28% of the patients and genotype 3 in 72%. The G allele frequency of the IL-6 −174C/G polymorphism was 72.8% in the HCV patients. No significant statistically differences were observed in the IL-6 allele and genotype frequencies between HCV patients and controls as well as among markers of HCV infection (viral load, fibrosis, inflammation and aminotransferases) and IL-6 genotypes. In conclusion, IL-6 −174C/G polymorphism is not associated with clinical characteristics of chronic hepatitis C in Brazilian patients.

Increased expression of interleukin-6 (IL-6) gene transcript in relation to IL-6 promoter hypomethylation in gingival tissue from patients with chronic periodontitis

ObjectiveDNA methylation of the cytokine genes may play a role in the pathogenesis of periodontitis. The aim of this study is to evaluate whether the alteration of interleukin-6 (IL-6) gene promoter methylation in the gingival tissue (GT) and peripheral blood (PB) is unique to chronic periodontitis (CP). DesignDNA isolated from the GT and PB of 25 patients with (CP) and 20 healthy controls (H) was modified with sodium bisulfite and analyzed for IL-6 promoter methylation with direct sequencing. The levels of IL-6 mRNA and serum IL-6 protein were evaluated by a quantitative reverse transcription polymerase chain reaction and an enzyme-linked immunosorbent assay. ResultsThe CP group showed that the overall methylation rates of IL-6 promoter that contained 19 cytosine-guanine dinucleotide (CpG) motifs were significantly decreased in GT in comparison to PB (p<0.001), which was significantly negatively correlated with the probing depth (p=0.003). The GT and PB of the H group displayed similar overall methylation rates. No significant difference was observed in the methylation rates at each CpG in GT in comparison to the PB in both groups. The levels of IL-6 mRNA in the GT and PB and serum IL-6 of the two groups were comparable. The ratio of IL-6 mRNA in the GT relative to the PB was significantly higher in the CP group than in the H group (p=0.03). ConclusionThe increased expression of IL-6 gene transcription may be related to IL-6 promoter hypomethylation in the GT from CP patients.

β-naphthoflavone interferes with cyp1c1, cox2 and IL-8 gene transcription and leukotriene B4 secretion in Atlantic cod (Gadus morhua) head kidney cells during inflammation.

The objective of this study was to evaluate how β-naphthoflavone interacts with lipopolysaccharide (LPS) and polyinosinic acid: polycytidylic acid (poly I: C) induced innate immune parameters as well as phase I and phase II detoxification enzymes in head kidney cells isolated from Atlantic cod. β-naphthoflavone is a pure agonist of aryl hydrocarbon receptor (AhR) while LPS and poly I: C are not. β-naphthoflavone was added to head kidney leukocytes alone or together with LPS or poly I: C and the responses were evaluated in terms of protein and gene expression. The results showed that β-naphthoflavone (25nM), with and without LPS, significantly induced cytochrome P450 (cyp1c) transcription in cod head kidney cells. β-naphthoflavone (100nM) in the presence of the virus mimic, poly I: C, also increased cyp1c1transcription. LPS induced cyp1c1, cyclooxygenase 2 (cox2), interleukin 1β (IL-1β), interleukin 6 (IL-6) and interleukin 8 (IL-8) transcription, genes that were not affected by the tested β-naphthoflavone concentrations alone. However, β-naphthoflavone (25 and 50nM) strengthened LPS induced cox2 and IL-8 transcription. Cod head kidney cells exposed to β-naphthoflavone concentrations ranging from 25 to 100nM, with and without LPS or poly I: C, expressed AhR protein. LPS or β-naphthoflavone (5-50nM) significantly induced leukotriene B4 (LTB4) secretion compared to control. In conclusion, this study suggests that β-naphthoflavone could interfere with LPS induced immune cell signaling in cod head kidney cells.

Inflammation: TET2: the terminator.

The epigenetic modifier TET2 is required to repress interleukin-6 gene transcription and resolve inflammation.

Transcriptional mechanisms and protein kinase signaling mediate organic dust induction of IL-8 expression in lung epithelial and THP-1 cells.

Exposure to the agricultural work environment is a risk factor for the development of respiratory symptoms and chronic lung diseases. Inflammation is an important contributor to the pathogenesis of tissue injury and disease. Cellular and molecular mechanisms mediating lung inflammatory responses to agricultural dust are not yet fully understood. We studied the effects of poultry dust extract on molecular regulation of interleukin-8 (IL-8), a proinflammatory cytokine, in A549 and Beas2B lung epithelial and THP-1 monocytic cells. Our findings indicate that poultry dust extract potently induces IL-8 levels by increasing IL-8 gene transcription without altering IL-8 mRNA stability. Increase in IL-8 promoter activity was due to enhanced binding of activator protein 1 and NF-κB. IL-8 induction was associated with protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) activation and inhibited by PKC and MAPK inhibitors. IL-8 increase was not inhibited by polymyxin B or l-nitroarginine methyl ester, indicating lack of involvement of lipopolysaccharide and nitric oxide in the induction. Lung epithelial and THP-1 cells share common mechanisms for induction of IL-8 levels. Our findings identify key roles for transcriptional mechanisms and protein kinase signaling pathways for IL-8 induction and provide insights into the mechanisms regulating lung inflammatory responses to organic dust exposure.

Increased TNF α, IL-6 and ErbB2 mRNA expression in peripheral blood leukocytes from breast cancer patients.

Obesity has been associated with increased incidence and mortality of breast cancer. The precise relation between obesity and breast cancer is yet to be determined, with few studies linking them with altered serum levels adipokines and inflammatory cytokines. The relevance of the expression of genes encoding for adipokines and inflammatory cytokines in the peripheral blood and their contribution to obesity and breast cancer has not been fully investigated. We aim to identify potential transcriptional biomarkers in blood samples that may assist to underpin the link between obesity and breast cancer. Therefore, have investigated whether or not the expression levels, of selected genes [tumor necrosis factor-α (TNFα), interleukin 6 (IL-6), adiponectin, leptin, C-reactive protein (CRP), parathyroid hormone (PTH), tumor protein 53 (TP53) and erythroblastic leukemia viral oncogene 2 (ErbB2)] were altered in blood samples of lean, overweight/obese and breast cancer subjects. Blood samples were obtained from 37 lean, 19 overweight/obese and 12 breast cancer patients. Real-time polymerase chain reaction assays were performed to detect TNFα, IL-6, adiponectin, leptin, CRP, PTH, TP53 and ErbB2 gene transcripts. Transcript levels of TNFα were significantly higher by 1.4-fold and 2.1-fold in blood cells of overweight/obese and breast cancer patients, respectively, compared with lean control subjects. Transcript levels of IL-6 were significantly higher by 2.3-fold in blood cells from breast cancer patients compared with lean control subjects with normal body mass index, and no significant difference was found in the expression level of IL-6 transcripts between overweight/obese and lean control subjects. The ErbB2 transcript levels were significantly higher by 4.72-fold compared to lean control subjects and were also significantly higher compared to overweight/obese subjects. Breast cancer and obesity are associated with altered mRNA levels of cytokines and tumor marker in peripheral blood.

LPS induces IL‐8 expression through TLR4, MyD88, NF‐kappaB and MAPK pathways in human dental pulp stem cells

To evaluate the effects of lipopolysaccharide (LPS) on interleukin-8 (IL-8) and related intracellular signalling pathways in human dental pulp stem cells (hDPSCs). Human pulp tissues were isolated from human impacted third molars, and the hDPSCs were cultured and characterized. The effects of LPS on IL-8 and Toll-like receptor 4 (TLR4) gene expression in hDPSCs were investigated using real-time quantitative RT-PCR and ELISA. Whether TLR4/MyD88/NF-кB was involved in the LPS-induced up-regulation of IL-8 in hDPSCs was determined using transient transfection, luciferase assay and ELISA. The involvement of MAPKs in the LPS-induced up-regulation of IL-8 in hDPSCs was investigated via transient transfection, luciferase assay, ELISA and western blot. The data were statistically analysed using Student's t-test or one-way anova followed by the Student-Neumann-Keuls test. Cells exposed to LPS not only displayed an enhanced expression of TLR4 but also showed an elevated IL-8 gene expression; exposure to LPS also resulted in the induction of IL-8 gene transcription via promoter activation. The LPS-induced IL-8 promoter activation was inhibited through dominant-negative mutations in TLR4 and MyD88, but not in TLR2. The LPS-induced IL-8 protein release was attenuated through the administration of TLR4-neutralizing antibody or MyD88 inhibitory peptide and a dominant-negative mutation in IκBα. In contrast, IL-8 protein release was enhanced through the expression of NF-κB p65. Treatment with PDTC, TPCK or Bay117082 effectively antagonized LPS-induced IL-8 protein release. Moreover, both the promoter activity and the LPS-induced release of IL-8 were diminished upon the administration of U0126 and SB203580, but not SP600125. Moreover, the exposure to LPS activated ERK1/2 and p38 MAPK phosphorylation in cells. This study reports the LPS-mediated transcriptional and post-translational up-regulation of IL-8, which is a process that also involves TLR4, MyD88, NF-κB and MAPK.

Contraction-induced Interleukin-6 Gene Transcription in Skeletal Muscle Is Regulated by c-Jun Terminal Kinase/Activator Protein-1

Exercise increases the expression of the prototypical myokine IL-6, but the precise mechanism by which this occurs has yet to be identified. To mimic exercise conditions, C2C12 myotubes were mechanically stimulated via electrical pulse stimulation (EPS). We compared the responses of EPS with the pharmacological Ca(2+) carrier calcimycin (A23187) because contraction induces marked increases in cytosolic Ca(2+) levels or the classical IκB kinase/NFκB inflammatory response elicited by H(2)O(2). We demonstrate that, unlike H(2)O(2)-stimulated increases in IL-6 mRNA, neither calcimycin- nor EPS-induced IL-6 mRNA expression is under the transcriptional control of NFκB. Rather, we show that EPS increased the phosphorylation of JNK and the reporter activity of the downstream transcription factor AP-1. Furthermore, JNK inhibition abolished the EPS-induced increase in IL-6 mRNA and protein expression. Finally, we observed an exercise-induced increase in both JNK phosphorylation and IL-6 mRNA expression in the skeletal muscles of mice after 30 min of treadmill running. Importantly, exercise did not increase IL-6 mRNA expression in skeletal muscle-specific JNK-deficient mice. These data identify a novel contraction-mediated transcriptional regulatory pathway for IL-6 in skeletal muscle.

The Myxobacterial Compounds Spirangien A and Spirangien M522 Are Potent Inhibitors of IL‐8 Expression

Elevated expression of interleukin-8 (IL-8) has been implicated in inflammatory diseases, in tumor growth, and in angiogenesis. The aim of this study was to identify natural or synthetic compounds that suppress IL-8 production in response to interleukin-1 (IL-1), the natural inflammatory stimulus of the IL-8 gene. We therefore developed an IL-1-inducible cell-based screening assay by stable integration of an IL-8 reporter gene into HeLa S3 cells. The screening of heterogeneous compound libraries revealed several compounds that displayed an inhibitory effect on the reporter gene expression. Following hit validation, we focused on the most efficient compound, spirangien A, and its chemical derivate spirangien M522. Detailed analysis shows that both compounds are potent inhibitors of the endogenous IL-8 gene transcription. Furthermore, both compounds decelerate the phosphorylation and degradation of IκBα, the key regulator of the IL-1-stimulated NF-κB signaling pathway. Our study has identified the two spirangiens A and M522 as potent inhibitors of IL-1/NF-κB-mediated IL-8 gene expression.

Effects of the interleukin-6 (IL-6) polymorphism on toxic metal and trace element levels in placental tissues

The placenta is a crucial organ of fetal origin that functions in providing nutrients to the fetus from the mother. During pregnancy, the need for essential micronutrients, such as Fe and Zn, increases due to the requirements of the growing fetus. Maternal Fe deficiency induces an increase in Cu levels and can also affect cytokine levels in the placenta. On the other hand, Cu deficiency, although not as common, can also have destructive effects on the fetus. Interleukin-6 (IL-6) is a pleiotropic cytokine with a wide range of biological activities, including such as immune responses, acute-phase reactions, and inflammation. The placenta produces a significant amount of IL-6 during pregnancy. The effects of the IL-6 −174 G/C single nucleotide polymorphism (SNP) on IL-6 gene transcription and on plasma cytokine levels were assessed in the present study. We investigated the association between the IL-6 −174 G/C polymorphism and trace element/toxic metal levels in placental tissues. For the purposes of this study, 95 healthy volunteers were evaluated. Presence of the IL-6 polymorphism was determined using the standard polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) technique, and metal levels were analyzed by atomic absorption spectrometry (AAS). Based on our data, there were no significant associations between the IL-6 −174 G/C polymorphism and Pb, Cd, Fe, or Zn levels in the placental tissues (p > 0.05), but a statistically significant association was detected between the polymorphism and Cu levels (p = 0.016). We determined that the mean Cu levels in the placental tissues from individuals with GG, GC and CC genotypes were 5.62 ± 1.98, 6.22 ± 3.22 and 8.00 ± 1.32 ppm, respectively, whereas the overall mean Cu level from the placental tissues was 5.98 ± 2.51 ppm.

Role of interleukin-6 −174 G/C promoter polymorphism in trace metal levels of autopsy kidney and liver tissues

Interleukin-6 (IL-6) gene is a multifunctional cytokine which is expressed in lymphocytes, fibroblasts, macrophages, in response to different types of inflammatory stimuli. IL-6 also controls induction and expression of metallothioneins (MTs) which maintain homeostasis of zinc and copper. In human, IL-6 gene is located on chromosome 7p21 and -174 G/C polymorphism located in its promoter region. Recently, genetic studies showed that IL-6 -174 G/C promoter polymorphism influences IL-6 gene transcription and plasma cytokine levels. The aim of this study is to determine the IL-6 promoter polymorphism effect on trace element levels and toxic metal accumulation in the kidney and liver tissues. Kidney and liver tissues were collected from 122 autopsy cases in Ankara district. IL-6 promoter polymorphism was detected by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) analysis. The genotype frequencies were found as 54.9% homozygote typical (GG), 39.3% heterozygote (GC) and 5.7% homozygote atypical (CC). The allele frequencies in all subjects were consistent with Hardy-Weinberg equilibrium (χ(2) = 0.179; p > 0.05). In order to assess the cadmium (Cd), lead (Pb), zinc (Zn) and copper (Cu) levels in the autopsy tissues, a dual atomic absorption spectrophotometer system was used. As a result, no statistical association was found between the IL-6 promoter polymorphism and Pb, Cd, and Cu (p > 0.05) levels in the kidney and liver tissues but statistically significant differences were detected with the Zn concentration (p < 0.05).

Integrin-linked kinase regulates melanoma angiogenesis by activating NF-κB/interleukin-6 signaling pathway

Integrin-linked kinase (ILK) is a highly conserved serine-threonine protein kinase involved in cell-extracellular matrix interactions, cytoskeletal organization and cell signaling. Overexpression of ILK in epithelial cells leads to anchorage-independent growth with increased cell cycle progression. Previously, we have shown that ILK upregulation strongly correlates with melanoma progression, invasion and inversely correlates with 5-year survival of melanoma patients. However, the molecular mechanism by which ILK enhances melanoma progression is currently unknown. In the present study, we found that proangiogenic molecule interleukin-6 (IL-6) is the downstream target of ILK in melanoma cells. ILK overexpression increased IL-6, whereas silencing of ILK suppressed IL-6 expression at both messenger RNA and protein levels. ILK also altered the activity and subcellular localization of nuclear factor-kappaB (NF-κB) subunit p65. We further found that ILK enhanced the IL-6 gene transcription by promoting the binding of NF-κB p65 to IL-6 promoter. Moreover, ILK overexpression in melanoma cells enhanced the tube-forming ability of endothelial cells in vitro and microvessel formation in vivo. ILK-induced tube and blood vessel formation of endothelial cells was significantly reduced upon IL-6 inhibition in ILK-overexpressing melanoma cells. To delineate the mechanism by which ILK-induced IL-6 production can enhance angiogenesis, further analysis of the downstream targets of IL-6 signaling showed an increased activity of the signal transducer and activator of transcription 3 (STAT3) in ILK-overexpressing cells. As STAT3 binds to vascular endothelial growth factor (VEGF) promoter, we found that VEGF levels were elevated in ILK-overexpressing cells and declined upon transfection of IL-6 small interfering RNA, suggesting that ILK may regulate VEGF expression through IL-6 pathway by activating STAT3.

Differential Effects of Multiplicity of Infection on Helicobacter pylori-Induced Signaling Pathways and Interleukin-8 Gene Transcription

Interleukin-8 (IL-8) plays a central role in the pathogenesis of Helicobacter pylori infection. We used four different H. pylori strains isolated from patients with gastritis or duodenal ulcer disease to examine their differential effects on signaling pathways and IL-8 gene response in gastric epithelial cells. IL-8 mRNA level is elevated in response to high (100) multiplicity of infection (MOI) independent of cagA, vacA, and dupA gene characteristics. By lower MOIs (1 or 10), only cagA ( + ) strains significantly induce IL-8 gene expression. This is based on differential regulation of IL-8 promoter activity. Analysis of intracellular signaling pathways indicates that H. pylori clinical isolates induce IL-8 gene transcription through NF-κB p65, but by a MOI-dependent differential activation of MAPK pathways. Thus, the major virulence factors of H. pylori CagA, VacA, and DupA might play a minor role in the level of IL-8 gene response to a high bacterial load.