Regulation Of IL-6 Expression Research Articles

Thyrotropin Regulates IL-6 Expression in CD34+ Fibrocytes: Clear Delineation of Its cAMP-Independent Actions

IL-6 plays diverse roles in normal and disease-associated immunity such as that associated with Graves’ disease (GD). In that syndrome, the orbit undergoes remodeling during a process known as thyroid-associated ophthalmopathy (TAO). Recently, CD34+ fibrocytes were found to infiltrate the orbit in TAO where they transition into CD34+ orbital fibroblasts. Surprisingly, fibrocytes display high levels of functional thyrotropin receptor (TSHR), the central antigen in GD. We report here that TSH and the pathogenic anti-TSHR antibodies that drive hyperthyroidism in GD induce IL-6 expression in fibrocytes and orbital fibroblasts. Unlike TSHR signaling in thyroid epithelium, that occurring in fibrocytes is completely independent of adenylate cyclase activation and cAMP generation. Instead TSH activates PDK1 and both AKT/PKB and PKC pathways. Expression and use of PKCβII switches to that of PKCµ as fibrocytes transition to TAO orbital fibroblasts. This shift is imposed by CD34− orbital fibroblasts but reverts when CD34+ fibroblasts are isolated. The up-regulation of IL-6 by TSH results from coordinately enhanced IL-6 gene promoter activity and increased IL-6 mRNA stability. TSH-dependent IL-6 expression requires activity at both CREB (−213 to −208 nt) and NF-κB (–78 to −62 nt) binding sites. These results provide novel insights into the molecular action of TSH and signaling downstream for TSHR in non-thyroid cells. Fibrocytes neither express adenylate cyclase nor generate cAMP and thus these findings are free from any influence of cAMP-related signaling. They identify potential therapeutic targets for TAO.

Critical role of histone deacetylase 11 in lineage differentiation of immune cells (P4378)

Abstract Previously our group reported HDAC11 as a regulator of IL-10 expression in immune cells; however the functions of HDAC11 still remain largely unknown. Here we reveal the novel role of HDAC11 as an epigenetic regulator of lineage differentiation in leukocytes. To “visualize” dynamic changes in HDAC11 transcriptional activation, we utilized HDAC11 promoter-driven eGFP reporter mice, where eGFP expression is indicative of HDAC11 transcription. Using this system, we found that hematopoietic stem cells are devoid of eGFP expression. However, eGFP expression increases from CD4-/CD8- T-cells to single positive CD4+ and CD8+, and decreases in effector compared to naïve T-cells. In the bone marrow, expression of eGFP moderately increases in the transition from Pro- to Pre-B-cells, however in the periphery eGFP significantly increases in the B-1 stage of differentiation. Remarkably, in plasma cells eGFP expression appears to be at its highest. Moreover, less plasma cells and more B-1 cells were observed when HDAC11KO were compared to wild-type mice. Expression of eGFP in myeloblasts appears to be absent, however it increases to 50% in the promyelocytes and to 98% in the granulocytes. Strikingly, monocytes showed no eGFP expression. These results unveiled a previously unknown role of HDAC11 in hematopoiesis, regulating leukocyte lineage differentiation and expansion. These key findings will potentially lead to targeted epigenetic therapies to influence immune cell differentiation.

Abstract 4194: The hereditary genetic polymorphism of mir-608 predicts the clinical outcome and expression of IL-6 in patients with esophageal squamous cell carcinoma .

Abstract PURPOSE: To investigate the effect of the single nucleotide polymorphisms (SNPs) in microRNA-related genes on the prognosis of patients with esophageal squamous cell carcinoma (ESCC). PATIENTS AND METHODS: A total of 504 patients with a diagnosis of ESCC were enrolled in the study. The genotypes of miRNA-related SNPs were analyzed from the genomic DNA of peripheral leukocytes and were correlated with the clinical outcome of patients, which was first evaluated in a randomly assigned training set (n=129) and subsequently verified by a replication set (n= 375). RESULTS: In training group, the heterozygous CG genotype of mir-608 rs4919510 was associated with reduced risk for adverse clinical outcomes (HR [95% CI] =0.47 [0.27-0.82] for death; HR [95% CI] =0.47 [0.29-0.77] for recurrence). In contrast, both homozygous CC and GG genotypes showed unfavorable for prognosis. The associations were confirmed in an independent replication group (GG+CC vs. CG, HR [95% CI] =1.36 [1.07-1.73], P=0.013 for death; HR [95% CI] = 1.26 [1.00-1.59], P=0.048 for recurrence). The prognostic effect was more pronounced among patients with advanced tumor stages and in those treated with concurrent neoadjuvant chemo- radiotherapy (CCRT). Functional relevance of rs4919510 was correlated with the serum IL-6 levels. GG variant was significantly associated with IL-6 over-expression in advanced ESCC patients. CONCLUSION: The hereditary genetic variants in mir-608 can serve as an independent predictor for the clinical outcome of ESCC patients. The prognostic effect of rs4919510 may be partially mediated by impairing the ability in regulating IL-6 expression. Citation Format: Pei-Wen Yang, Ching-Yueh Hsieh, Yu-Ting Huang, Ya-Chuan Huang, Tzu-Hsuan Chiang, Jang-Ming Lee. The hereditary genetic polymorphism of mir-608 predicts the clinical outcome and expression of IL-6 in patients with esophageal squamous cell carcinoma . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4194. doi:10.1158/1538-7445.AM2013-4194

Characterization of contraction-induced IL-6 up-regulation using contractile C2C12 myotubes

Muscle contractile activity functions as a potent stimulus for acute interleukin (IL)-6 expression in working skeletal muscles. Recently, we established an "in vitro contraction model" using highly-developed contractile C2C12 myotubes by applying electric pulse stimulation (EPS). Herein, we characterize the effects of EPS-evoked contraction on IL-6 expression in contractile C2C12 myotubes. Both secretion and mRNA expression of IL-6 were significantly up-regulated by EPS in a frequency-dependent manner in contracting myotubes during a 24-h period, and the response was blunted by cyclosporine A, a calcineurin inhibitor. Longer time (~12h) was required for the induction of IL-6 after the initiation of EPS as compared to that of other contraction-inducible CXC chemokines such as CXCL1/KC, which were induced in less than 3 hours. Furthermore, these acute inducible CXC chemokines exhibited no autocrine effect on IL-6 expression. Importantly, contraction-dependent IL-6 up-regulation was markedly suppressed in the presence of high levels of glucose along with increased glycogen accumulations. Experimental manipulation of intracellular glycogen contents by modulating available glucose or pyruvate during a certain EPS period further established the suppressive effect of glycogen accumulations on contraction-induced IL-6 up-regulation, which appeared to be independent of calcineurin activity. We also document that EPS-evoked contractile activity improved insulin-responsiveness in terms of intracellular glycogen accumulations. Taken together, these data provide important insights into the regulation of IL-6 expression in response to contractile activity of muscle cells, which is difficult to examine using in vivo experimental techniques. Our present results thus expand the usefulness of our "in vitro contraction model".

Negative Regulation of IL-10 Expression in CD4 T Cells by Growth Factor Independent-1

Negative Regulation of IL-10 Expression in CD4 T Cells by Growth Factor Independent-1

Transcriptional Regulation of IL-10 Expression by Peroxisome Proliferator-Activated Receptor (Pparγ) in Human Intestinal Epithelial Cells

IL-10 is an important immunomodulatory cytokine in the regulation of intestinal inflammation in inflammatory bowel disease (IBD). PPARγ is a nuclear receptor known to modulate transcriptional activities of many cytokines and is highly expressed in intestinal epithelial cells (IECs). Human IECs have been shown to express IL-10, but how its expression is regulated remains obscure especially in the setting of inflammation. We have demonstrated that intestinal 15d-PGJ2 (an endogenous PPARγ ligand) is predominantly induced by infiltrated macrophages (Mφs) through toll-like receptor 4 (TLR4)-mediated Cox-2 induction during colitis. In this study, we addressed whether PPARγ activation regulates expression of IL-10 in IECs. Human IEC cell line SW480-APC expressing TLR4 (top) and mouse peritoneal Mφs (bottom) were co-cultured in transwells. 15d-PGJ2 production and IL-10 mRNA expression were measured by enzyme linked assay and real-time PCR in the presence or absence of lipopolysaccharide (LPS). In order to examine whether the IL-10 promoter has PPARγ responsive elements, a 2,000 bp of IL-10 promoter was cloned from SW480-APC genomic DNA into the luciferase reporter vector. Expression and activity of PPARγ were blocked by siRNA and a specific inhibitor SR202, respectively. The IL-10 promoter activity was analyzed by luciferase reporter assay. PPARγ activity was confirmed using a reporter vector PPRE X3-TK-luc. Co-culturing SW480-APC with Mφs increased IL-10 expression in IECs within 24 hours, which was significantly enhanced by LPS stimulation in later time point (72 hours). LPS stimulation of SW480-APC resulted in PPARγ activation peaked at 48 hours. During SW480-APC/Mφ co-culture, LPS stimulation induced 15d-PGJ2 production, which is abolished when TLR4-deficient Mφs were used. Knocking down the expression of PPARγ suppressed IL-10 promoter activity and mRNA expression in SW480-APC in baseline as well as after LPS stimulation. These results suggest that PPARγ positively regulates IL-10 expression in IECs at the transcriptional level. During inflammation, mucosal Mφs induce PPARγ ligand (15d-PGJ2) in a TLR4-dependent manner that contributes to PPARγ activation in IECs and thus IL-10 expression. This may be an intrinsic immunoregulatory program that is mediated through host-bacterial interactions, which dampens on-going inflammation during intestinal inflammation and thus can be a novel therapeutic target for IBD.

The role of T-bet in obesity: lack of T-bet causes obesity in male mice

The association of T helper (Th) 1 cells with obesity is well documented in both animals and humans. The T-box transcription factor (T-bet) is known as the transcription factor that is responsible for the development of Th1 cells. However, the role of T-bet in obesity has never been elucidated. The present study aimed to investigate the regulatory function of T-bet on obesity in mice. Th1 cytokine levels were decreased, whereas Th2 cytokine level and GATA-3 messenger RNA (mRNA) expression were increased in T-bet knockout (KO) mice. T-bet KO male mice induced obesity as a result of increased body weight and food efficiency despite the fact that they feed a control diet. T-bet KO mice have an increase in weight of white adipose tissue and levels of triacylglyceride and low-density lipoprotein cholesterol. Interestingly, the expression levels of energy expenditure-related genes were decreased in T-bet KO mice. Both T-bet KO male and female mice had impaired glucose tolerance. In white adipose tissue, leptin, the increase in peroxisome proliferator receptor-γ and CAAT/enhancer-binding protein α mRNA expressions in T-bet KO mice was more than that in wild-type mice. Furthermore, we found that the level of interleukin (IL)-6 mRNA expression in white adipose tissue was elevated in T-bet KO mice but not IL-1β and tumor necrosis factor-α. IL-6 mRNA expression was increased in adipocyte fraction and stromal vascular fraction in white adipose tissue of T-bet KO mice. Taken together, our results reveal that T-bet may affect obesity through the regulation of IL-6 expression in adipocytes of white adipose tissue.

Altered let-7 expression in Myasthenia gravis and let-7c mediated regulation of IL-10 by directly targeting IL-10 in Jurkat cells

Myasthenia gravis (MG) is a T cell-dependent and B cell-mediated autoimmune disease of neuromuscular junctions and cytokines may play a crucial role in the pathogenesis and perpetuation of MG. MicroRNAs (miRNAs) have been implicated as fine-tuning regulators controlling diverse biological processes at the level of posttranscriptional repression. Dysregulation of miRNAs has been described in various disease states. In this study, miRNA microarrays identified let-7 family to be decreased in peripheral blood mononuclear cells (PBMCs) from MG patients compared to the healthy controls. We next demonstrated the differential expression of let-7 family in larger samples by quantitative real-time PCR. Using a combination of bioinformatics and molecular approaches, we confirmed IL-10 as a target for let-7c. IL-10 expression also showed a negative correlation with let-7c expression in PBMCs from MG patients. Further experiments revealed that induced levels of IL-10 were inversely related to let-7c levels. We also showed that let-7c could regulate IL-10 expression in Jurkat cells. In summary, our results suggest that abnormal expression/regulation of microRNAs may contribute to or be indicative of the initiation and progression of MG.

TLR9- and Src-dependent expression of Krueppel-like factor 4 controls interleukin-10 expression in pneumonia

The release of potent pro-inflammatory mediators is crucial to mounting an efficient host response during infection. However, excessive inflammation may lead to deleterious tissue damage. This is highlighted in severe pneumococcal pneumonia, in which the delicate balance between a robust inflammatory response necessary to kill pneumococci and the loss of organ function determines the outcome of the disease. We assessed the regulation of the potent anti-inflammatory cytokine interleukin (IL)-10 in pneumococcal infection via Western blot, ELISA and chromatin immunoprecipitation analysis. Streptococcus pneumoniae induced IL-10 expression in mouse lungs and human lung epithelial cells. Pneumococcal infection resulted in a strong induction of Krueppel-like factor (KLF)4 expression in vivo and in vitro. The induction of both IL-10 and KLF4 is mediated by a pathway involving bacterial DNA, Toll-like receptor (TLR)9, MyD88 and Src kinase. KLF4 is recruited to the il10 promoter, and small-interfering RNA-mediated knockdown of KLF4 expression blocked IL-10 expression during pneumococcal infection. In conclusion, KLF4 is induced in a bacterial DNA-TLR9-Src-dependent manner and regulates IL-10 expression, linking the detection of bacterial DNA by TLR9 to the control of an inflammatory response.

Neuromedin B and Its Receptor Influence the Activity of Myometrial Primary Cells In Vitro Through Regulation of Il6 Expression via the Rela/p65 Pathway in Mice1

The neuromedin B receptor (Nmbr) is an important physiological regulator of spontaneous activities and stress responses through different cascades as well as its autocrine and paracrine effects. Previous studies have revealed that neuromedin B (Nmb) and its receptor signal via the Rela (also known as p65)/Il6 pathway in a mouse model of pregnancy. This study investigated the mechanism of Nmbr signaling via the Rela/p65-Il6 pathway and regulation of the concentration of intracellular free calcium ([Ca(2+)](i)) during the onset of labor in primary mouse myometrial cell cultures isolated from mice in term labor. Data demonstrated Nmbr agonist-mediated upregulation of the DNA binding activity of Rela/p65, Il6 expression, and [Ca(2+)](i) in a concentration-dependent manner. Furthermore, a significant correlation was observed between DNA binding activity of Rela/p65 and Il6 expression. Moreover, this up-regulation was blocked by Nmbr and Rela/p65 knockdown, achieved by RNA interference (RNAi) technology. No significant differences were identified in the inhibition of Il6 expression as a result of Nmbr or Rela/p65 knockdown. However, significant differences were observed between the [Ca(2+)](i) in Rela/p65-specific group and that in the Nmbr-specific small interfering RNA (siRNA)-treated groups. These data demonstrated that the Nmb/Nmbr interaction in pregnant myometrial primary cells in vitro predominantly influenced uterine activity through regulation of Il6 expression via the Rela/p65 pathway, although the effects of Nmbr on [Ca(2+)](i) involved several pathways that remain to be elucidated.

MiR-146a regulated IL-6 expression promotes monocyte proliferation in sepsis patients (117.22)

Abstract Sepsis is a serious medical condition, which is characterized by a systemic inflammatory state induced by a known or suspected infection. Despite extensive basic research and clinical studies, the pathogenesis of sepsis is still poorly understood. In the current study, we found that total peripheral blood mononuclear cell counts increased significantly, however, lymphocytes including CD4+ and CD8+ T cells decreased significantly in sepsis patients. Further studies demonstrated that monocytes increased significantly in sepsis patients. Plasma levels of IL-6, IL-8 and MCP-1 dramatically increased, whereas plasma levels of RANTES significantly decreased in sepsis patients compared with normal controls. MiRNAs such as miR-143, miR-145, miR-146a, miR-181a, miR-182, miR-199a, miR-486 and miR-1308 were dysregulated in sepsis patients. Down-regulation of miR-146a may be responsible for the increased levels of IL-6 expression, and thus may promote monocyte proliferation in sepsis patients. These results suggest that the modulation of IL-6 expression by dysregulated miR-146a in the control of monocyte proliferation may have implications in the pathogenesis and treatment of sepsis.

Direct regulation of Interleukin-6 expression by Notch signaling in macrophages (172.36)

Abstract Notch signaling is required for immune cell development. The intracellular domain of Notch (NIC) induces expression of target genes by binding to CSL, a DNA-binding protein. In macrophages, Notch signaling regulates inflammatory cytokines TNFα and IL-6. IL-6 is secreted by various cell types, including activated macrophages and functions in immune regulation and inflammation. The consensus CSL binding site in the promoter region of IL-6 was shown to negatively regulate IL-6 mRNA, but the role of Notch signaling in IL-6 expression in macrophages remained unclear. We investigated the role of Notch signaling in the regulation of the IL-6 transcript in macrophages. Our results showed that bone marrow derived macrophages (BMMΦ) and RAW264.7 cell lines activated with lipopolysaccharide (LPS) and interferon gamma (IFNγ) induced IL-6 mRNA expression and upregulation of Notch1. Inhibition of Notch signaling in BMMΦ using a gamma secretase inhibitor partially decreases the level of IL-6 mRNA and protein. In contrast, RAW264.7, transiently transfected with constitutively active Notch1, displayed a dramatic increase in IL-6 mRNA. Inhibition of NF-κB completely abrogated IL-6 mRNA expression induced by the over expression of NIC. Chromatin immunoprecipitation assay demonstrated that Notch1 binds to the promoter of IL-6 in activated RAW264.7 but not in unstimulated cells. These results strongly suggest that Notch1 positively regulates IL-6 expression via NF-κB in activated macrophages.

Knockdown of aberrantly expressed nuclear localized decorin attenuates tumour angiogenesis related mediators in oral cancer progression model in vitro

BackgroundOral cancer accounts for roughly 3% of cancer cases in the world with about 350,000 newly reported cases annually and a 5-year survival rate of only 50%. Majority of oral cancers are squamous cell carcinomas that originate in the oral mucosal epithelial linings. We have previously shown that in human malignant squamous cells carcinoma (SCC-25) as well as in dysplastic oral keratinocytes (DOK), a small leucine-rich multifunctional proteoglycan decorin is aberrantly expressed and localized in the nucleus where it interacts with nuclear epidermal growth factor receptor (EGFR). Post-transcriptional silencing of nuclear decorin significantly reduced IL-8 and IL8-dependent migration and invasion in these dysplastic and malignant oral epithelia. The objective of this study was to further examine the effects of nuclear decorin silencing on angiogenesis and angiogenesis related mediators in this oral cancer progression cell line model.MethodsWe have used multiplex PCR, western blotting, and in vitro endothelial tube formation assay to study angiogenesis and related pathways in nuclear decorin silenced (stable knockdown) DOK and SCC-25 cells.ResultsNuclear decorin knockdown resulted in significant down regulation of IL-8 expression, however IL-10, and TGF-β expression was not affected in either DOK or SCC25 cells as measured by multiplex RT PCR. IL-8 receptor CXCR 1 and 2 expression was slightly lower in nuclear decorin silenced cells indicating a contributing mechanism in previously shown reduced IL-8 mediated migration and invasion phenotype in these cells. IL-8 is known to induce Matrix metalloproteinase 9 (MMP9) which not only plays a role in tumour migration and invasion but also induces angiogenic switch. We found MMP9 to be significantly reduced in nuclear decorin silenced dysplastic and malignant oral epithelia. Other potent angiogenic mediators, VEGF189 and ANG-1 were either significantly reduced or completely abrogated in these cells. Angiogenesis as measured by endothelial tube-like formations of HUVEC cells was reduced by almost 50 percent when HUVECs were incubated in the presence of conditioned medium form nuclear decorin silenced dysplastic and malignant cell lines as compared to respective controls.ConclusionsTogether these results indicate that aberrantly expressed nuclear localized decorin strongly influences angiogenic potential of dysplastic and malignant oral epithelial cells.

Different signaling mechanisms regulating IL-6 expression by LPS between gingival fibroblasts and mononuclear cells: seeking the common target

To reduce connective tissue IL-6 level stimulated by LPS, it is essential to control IL-6 expression in both mononuclear cells and fibroblasts. However, it is unclear whether the regulatory mechanisms for both cells are similar or not. In this study, we found that signaling pathways mediating LPS-stimulated IL-6 in mononuclear U937 cells and fibroblasts were different. Furthermore, our studies showed that while LPS activated AP-1 and NFκB in U937 cells, it only activated NFκB in fibroblasts. Analysis of nuclear AP-1 subunits showed that LPS stimulated c-Fos, Fra-1 and Jun D activities in U937 cells, but not fibroblasts. The lack of ERK involvement in LPS-stimulated IL-6 in fibroblasts was further supported by the observations that simvastatin, which is known to target ERK-AP-1, failed to inhibit LPS-stimulated IL-6 by fibroblasts. Finally, we showed that targeting NFκB pathway was highly effective in inhibition of LPS-stimulated IL-6 in coculture of U937 cells and fibroblasts.

Myeloid Cell IL-10 Production in Response to Leishmania Involves Inactivation of Glycogen Synthase Kinase-3β Downstream of Phosphatidylinositol-3 Kinase

Leishmania disease expression has been linked to IL-10. In this study, we investigated the regulation of IL-10 production by macrophages infected with Leishmania donovani. Infection of either murine or human macrophages brought about selective phosphorylation of Akt-2 in a PI3K-dependent manner. These events were linked to phosphorylation and inactivation of glycogen synthase kinase-3β (GSK-3β) at serine 9, as the latter was abrogated by inhibition of either PI3K or Akt. One of the transcription factors that is negatively regulated by GSK-3β is CREB, which itself positively regulates IL-10 expression. Infection of macrophages with leishmania induced phosphorylation of CREB at serine 133, and this was associated with enhanced CREB DNA binding activity and induction of IL-10. Similar to phosphorylation of GSK-3β, both phosphorylation of CREB at serine 133 and CREB DNA binding activity were abrogated in cells treated with inhibitors of either PI3K or Akt prior to infection. Furthermore, disruption of this pathway either by inhibition of Akt or by overexpression of GSK-3β markedly attenuated IL-10 production in response to leishmania. Thus, GSK-3β negatively regulates myeloid cell IL-10 production in response to leishmania. Switching off GSK-3β promotes disease pathogenesis.

Interleukin-10 is expressed in HepG2.2.15 cells and regulated by STAT1 pathway

This study investigated the expression profiles of IL-10 gene in three human hepatoma cell lines including Huh7, HepG2, and HepG2 transfected with a plasmid containing hepatitis B virus (HBV) named HepG2.2.15. RT-PCR analysis demonstrated that IL-10 message RNA was absent in HepG2 and Huh7 cells, whereas it was present in HepG2.2.15 cells, which was consistent with ELISA result. Furthermore, except for lamivudine other antiviral treatments did not significantly decrease the HBV DNA level in HepG2.2.15 cells, while they had different effects on the expression of IL-10 protein, although stimulation by LPS had no significant effect. In addition, except for poly(I:C), the other treatments decreased the expression of IL-10 protein to different degrees, but had no significant effects on the expression of NF-κB and MyD88. Meanwhile, all treatments we used had effect on the expression of STAT1. In conclusion, IL-10 was expressed in HepG2.2.15 cells and STAT1 pathway might be involved in the regulation of IL-10 expression in HepG2.2.15 cells, but it was not the sole pathway, the exact mechanism warrants further study.

MIR-200c regulates IL-8 expression in leiomyoma by targeting IKKβ expression and NFκB activity

Enhanced inflammatory condition and aberrant expression of miRNAs has been associated with tumorigenesis and cellular transformation. The objective of this study was to assess the regulatory function of miR-200c in the above processes in leiomyomas (LYO) as compared to paired myometrium (MYO).

Colitis Development in IL-10 Deficient Mice Reveals a Direct Role of MCH in Regulating IL-10 Expression by Monocytes

Colitis Development in IL-10 Deficient Mice Reveals a Direct Role of MCH in Regulating IL-10 Expression by Monocytes

Abstract 402: The role of IL-6 in communication of pancreatic carcinoma cells with fibroblasts

Abstract <INTRODUCTION> Pancreatic cancer shows high mortality rates in gastroenterogical neoplasms, and there are difficulties in early diagnosis and aggressive progression. IL-6 has pleiotropic function and are produced by various immunnocompetent cells, as well as cancer cells. Some studies have been demonstrated IL-6 play an important role in evading host immune surveillance in tumor microenvironment, but interactions of fibroblasts has not been fully understood. Therefore, the aim of this study is to reveal role of fibroblasts in pancreatic tumor microenvironment. Especially, we evaluate induction and biologicial function of IL-6 in pancreatic carcinoma cells after the stimulation with various cytokines and fibroblast associated factors. <METHODS and RESULTS> Firstly, we examined IL-6 and its receptors expression by RT-PCR and Northern blotting. All 7 tested cell lines expressed gp130 and IL-6R alpha mRNA, 2 cell lines (Hs7667 and Capan1) expressed IL-6 mRNA in serum free condition. To clarify the effect of proinflammatory cytokines on IL-6 expression in Hs766T cells, cells were stimulated with or without cytokines. Northern blotting revealed TNFα and IL-1β upregulated IL-6 mRNA, but not IL-6, IL-8 and LIF. IL-6 did not affect cell proliferation by WTS assay in 7 pancreatic carcinoma cells. In contrast, IL-6 promoted cell motility significantly by gold particles phagocytosis assay (P<0.05) and cell invasion significantly by matrigel insert chamber assay (P<0.05). IL-6 induces phosphorylation of STAT3 in Hs-766T cells, but not AKT. To identify IL-6 expression by interaction between pancreatic carcinoma cells and fibroblasts, we used two established fibroblastic cell lines (MRC-9 and WI-38) isolated from human embryonal lung tissues. Serum free conditioned medium (CM) were collected after incubation for indicated periods. Hs766T originated CM (Hs766T-CM) induced IL-6 and IL-8 mRNA in MRC-9 and WI-38 cells. MRC-9 CM and WI-38-CM did not affect in Hs-766T cells. Co-culture between Hs-766T and MRC-9 cells induced IL-8 mRNA dratically. IL-6 promotes miR-146a and miR155 expression in Hs-766T cells. <CONCLUSIONS> Communication of pancreatic carcinoma cells with fibroblasts affect IL-6 expression and it could contribute to pancreatic cancer progression. Regulation of IL-6 expression in tumor microenvironment would be important for pancreatic cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 402. doi:10.1158/1538-7445.AM2011-402

Dragon (Repulsive Guidance Molecule b) Inhibits IL-6 Expression in Macrophages

Repulsive guidance molecule (RGM) family members RGMa, RGMb/Dragon, and RGMc/hemojuvelin were found recently to act as bone morphogenetic protein (BMP) coreceptors that enhance BMP signaling activity. Although our previous studies have shown that hemojuvelin regulates hepcidin expression and iron metabolism through the BMP pathway, the role of the BMP signaling mediated by Dragon remains largely unknown. We have shown previously that Dragon is expressed in neural cells, germ cells, and renal epithelial cells. In this study, we demonstrate that Dragon is highly expressed in macrophages. Studies with RAW264.7 and J774 macrophage cell lines reveal that Dragon negatively regulates IL-6 expression in a BMP ligand-dependent manner via the p38 MAPK and Erk1/2 pathways but not the Smad1/5/8 pathway. We also generated Dragon knockout mice and found that IL-6 is upregulated in macrophages and dendritic cells derived from whole lung tissue of these mice compared with that in respective cells derived from wild-type littermates. These results indicate that Dragon is an important negative regulator of IL-6 expression in immune cells and that Dragon-deficient mice may be a useful model for studying immune and inflammatory disorders.