Interleukin-6 Response Element Research Articles

Lipoprotein(a) and Its Potential Association with Thrombosis and Inflammation in COVID-19: a Testable Hypothesis.

Purpose of ReviewThe COVID-19 pandemic has infected over > 11 million as of today people worldwide and is associated with significant cardiovascular manifestations, particularly in subjects with preexisting comorbidities and cardiovascular risk factors. Recently, a predisposition for arterial and venous thromboses has been reported in COVID-19 infection. We hypothesize that besides conventional risk factors, subjects with elevated lipoprotein(a) (Lp(a)) may have a particularly high risk of developing cardiovascular complications.Recent FindingsThe Lp(a) molecule has the propensity for inhibiting endogenous fibrinolysis through its apolipoprotein(a) component and for enhancing proinflammatory effects such as through its content of oxidized phospholipids. The LPA gene contains an interleukin-6 (IL-6) response element that may induce an acute phase–type increase in Lp(a) levels following a cytokine storm from COVID-19.SummaryThus, subjects with either baseline elevated Lp(a) or those who have an increase following COVID-19 infection, or both, may be at very high risk of developing thromboses. Elevated Lp(a) may also lead to acute destabilization of preexisting but quiescent atherosclerotic plaques, which might induce acute myocardial infarction and stroke. Ongoing studies with IL-6 antagonists may be informative in understanding this relationship, and registries are being initiated to measure Lp(a) in subjects infected with COVID-19. If indeed an association is suggestive of being causal, consideration can be given to systematic testing of Lp(a) and prophylactic systemic anticoagulation in infected inpatients. Therapeutic lipid apheresis and pharmacotherapy for the reduction of Lp(a) levels may minimize thrombogenic potential and proinflammatory effects. We propose studies to test the hypothesis that Lp(a) may contribute to cardiovascular complications of COVID-19.

Abstract A42: Interleukin-6 induces secretion of SPINK1 and trypsin in colorectal cancer

Abstract Inflammation is known to promote colorectal cancer (CRC) tumorigenesis, but the underlying molecular mechanisms are still being uncovered. Proinflammatory cytokine interleukin-6 (IL-6) is primarily secreted by the cells of the tumor microenvironment (TME), mainly by inflammatory cells but also by activated fibroblasts. IL-6 stimulates growth and survival signaling in CRC. Inflammatory signals regulate also the production and activity of proteases and their inhibitors. Serine protease inhibitor Kazal type1 (SPINK1, aka tumor-associated trypsin inhibitor, TATI) is expressed in several tissues and over-expression of SPINK1 predicts an unfavorable outcome in many cancers, including colon cancer. The SPINK1 gene contains an IL-6 responsive element and in addition to being a protease inhibitor, it also acts as an acute phase reactant and a growth factor. Expression of serine proteases trypsin-1 and -2, the main targets of SPINK1, also correlate with malignancy and metastatic potential. The aim of this study was to assess the paracrine signaling between fibroblast-derived IL-6 and cancer cell-derived SPINK1, trypsin-1 and -2. The following expression analyses were used: quantitative PCR, immunohistochemistry, immunofluorometric assays and Western blotting. The results show that CRC cells Colo205 and HT-29 express SPINK1 and secrete it into the culture medium. IL-6 dose-dependently increased the mRNA expression and protein levels of SPINK1. Conditioned media from fibroblasts had the same effect, and conversely CRC media increased IL-6 secretion in the fibroblasts, indicating paracrine signaling between these cell types. In CRC tissues cancer cells were positive for SPINK1, while IL-6 was found in fibroblasts surrounding the cancer cells, confirming the in vitro results. In Colo205 cells the baseline levels of trypsin-1 and -2 and the respective genes PRSS1 and PRSS2 were much higher compared to HT-29 cells. In Colo205 cells IL-6 led to concomitant increase in the secretion of trypsin-1 and -2, whereas in HT-29 cells these remained constantly low. Mechanistically, addition of IL-6 led to activation of the canonical Stat3 pathway, as indicated by Stat3 phosphorylation. Stat3 inhibitor reduced both SPINK1 and trypsin-1 and -2 levels, demonstrating that Stat3 is the transcription factor driving their expression. Taken together, our results show a connection between TME-derived inflammatory response and increased SPINK1 and trypsin-1 and -2 levels. Elevated serum SPINK1 has been shown to be an independent prognostic factor in colon cancer. As SPINK1 acts as a growth factor in some cancers, it has also been suggested as a therapeutic target. Therefore assessment of SPINK1 expression and function has several potentially important clinical applications in colorectal and other cancers. The study also strengthens the role of tumor microenvironment in tumor progression and emphasizes the importance of studying tumor-stroma crosstalk of proteolytic processing. Citation Format: Kati Räsänen, Elina Lehtinen, Kristiina Nokelainen, Laura Hautala, Outi Itkonen, Ulf-Håkan Stenman, Hannu Koistinen. Interleukin-6 induces secretion of SPINK1 and trypsin in colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A42.

Interleukin-6 increases expression of serine protease inhibitor Kazal type 1 through STAT3 in colorectal adenocarcinoma.

Inflammation promotes colorectal cancer (CRC) tumorigenesis, but the underlying molecular mechanisms are still being uncovered. Proinflammatory cytokine interleukin-6 (IL-6) stimulates survival signaling in CRC; inflammatory signals also regulate production and activity of proteases and their inhibitors. Over-expression of serine protease inhibitor Kazal type 1 (SPINK1) predicts an unfavorable outcome in colon cancer. The SPINK1 gene contains an IL-6 responsive element, suggesting it could act as an acute phase reactant. We assessed the connection between IL-6 and SPINK1, and the function and mechanism of this signaling. Our results show that Colo205 and HT-29 cells express and secrete SPINK1, and both fibroblast-derived and recombinant IL-6 further increased the SPINK1 levels. Concurrently CRC cells augmented the IL-6 production in fibroblasts. In CRC tissues cancer cells were positive for SPINK1, whereas IL-6 was found in stromal cells. In Colo205 cells IL-6 also stimulated the secretion of trypsin-1 and -2, the key targets of SPINK1 protease inhibition, whereas in HT-29 cells trypsin-1 and -2 levels remained constantly low. Functionally, both IL-6 and SPINK1 increased the motility of the CRC cells. Mechanistically, IL-6 activated the canonical STAT3 pathway and inhibition of STAT3 phosphorylation decreased the levels of SPINK1, trypsin-1 and -2. Taken together, our results indicate a novel link between inflammatory signals originating from the tumor microenvironment and increased SPINK1 levels. This finding has potential therapeutic implications for targeted therapy, as it confirms that SPINK1 acts as an acute phase reactant and that it participates in the paracrine crosstalk with the tumor microenvironment of colon cancer. © 2015 Wiley Periodicals, Inc.

Abstract 439: Interleukin-6 induces SPINK1 in colorectal cancer

Abstract Inflammation is known to promote colorectal cancer (CRC) tumorigenesis, but the underlying molecular mechanisms are still being uncovered. Proinflammatory cytokine interleukin-6 (IL-6) stimulates growth and survival signaling in CRC cells. Inflammatory signals regulate also the production and activity of proteases and their inhibitors. Serine protease inhibitor Kazal type1 (SPINK1, aka tumor-associated trypsin inhibitor, TATI) is expressed in several tissues and over-expression of SPINK1 predicts an unfavorable outcome in many cancers, including colon cancer. The SPINK1 gene contains an IL-6 responsive element and in addition to being a protease inhibitor, it also acts as an acute phase reactant and a growth factor. The aim of this study was to assess the connection between IL-6 and SPINK1 and the effect of this signaling to growth and invasion of CRC cells. The following expression analyses and functional assays were used: quantitative PCR, immunohistochemistry, immunofluorometric assays, Western blotting, cell proliferation and migration assays. The results of this study show that CRC cell lines Colo205 and HT-29 express SPINK1 and secrete it into the culture medium. In Colo205 and HT-29 cells IL-6 dose-dependently increased the mRNA expression and protein levels of SPINK1. The baseline secretion was lower in the Colo205 cells, and significantly more IL-6 was required for the dose-dependent induction of SPINK1 expression in these cells as compared to HT-29 cells. Interestingly, in Colo205 the baseline levels of secreted tumor-associated trypsinogen-1 and -2 (TAT-1 and -2), the key targets of SPINK1 protease inhibitory action, were much higher than in HT-29 cells. In these cells IL-6 led to concomitant increase in the secretion of TAT-1 and -2, whereas in HT-29 cells the TAT-1 and -2 levels remained constantly low. IL-6 at concentrations giving the maximum SPINK1 induction did not increase the proliferation of Colo205 or HT-29 cells; however the motility of these cells was increased in response to this proinflammatory cytokine. Mechanistically, addition of IL-6 led to activation of the canonical signal transducer and activator of transcription 3 (Stat3) pathway, as indicated by Stat3 phosphorylation in both Colo205 and HT-29 cells. Taken together, our results indicate a connection between inflammatory response and increased SPINK1 levels. Elevated serum SPINK1 has been shown to be an independent prognostic factor in colon cancer. As SPINK1 acts as a growth factor in some cancers, it has also been suggested as a therapeutic target. Therefore assessment of SPINK1 expression and function has several potentially important clinical applications in colorectal and other cancers. Citation Format: Kati A. Räsänen, Laura Hautala, Ulf-Håkan Stenman, Hannu Koistinen. Interleukin-6 induces SPINK1 in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 439. doi:10.1158/1538-7445.AM2015-439

Nuclear matrix protein SMAR1 control regulatory T-cell fate during inflammatory bowel disease (IBD).

Regulatory T (Treg) cells are essential for self-tolerance and immune homeostasis. Transcription factor Foxp3, a positive regulator of Treg cell differentiation, has been studied to some extent. Signal transducer and activator of transcription factor 3 (STAT3) is known to negatively regulate Foxp3. It is not clear how STAT3 is regulated during Treg differentiation. We show that SMAR1, a known transcription factor and tumor suppressor, is directly involved in maintaining Treg cell fate decision. T-cell-specific conditional knockdown of SMAR1 exhibits increased susceptibility towards inflammatory disorders, such as colitis. The suppressive function of Treg cells is compromised in the absence of SMAR1 leading to increased T helper type 17 (Th17) differentiation and inflammation. Compared with wild-type, the SMAR1−/− Treg cells showed increased susceptibility of inflammatory bowel disease in Rag1−/− mice, indicating the role of SMAR1 in compromising Treg cell differentiation resulting in severe colitis. We show that SMAR1 negatively regulate STAT3 expression favoring Foxp3 expression and Treg cell differentiation. SMAR1 binds to the MAR element of STAT3 promoter, present adjacent to interleukin-6 response elements. Thus Foxp3, a major driver of Treg cell differentiation, is regulated by SMAR1 via STAT3 and a fine-tune balance between Treg and Th17 phenotype is maintained.

Hepatitis C Virus Proteins Inhibit C3 Complement Production

The third component of human complement (C3) plays a central role in innate immune function as its activation is required to trigger classical as well as alternative complement pathways. In this study, we have observed that sera from patients chronically infected with hepatitis C virus (HCV) displayed significantly lower C3 levels than sera from healthy individuals. Liver biopsy specimens from the same patients also exhibited lower C3 mRNA expression than liver tissues from healthy donors. C3 mRNA level was reduced in hepatocytes upon infection with cell culture-grown HCV genotype 1a or 2a in vitro. Further analysis suggested that HCV core protein displayed a weak repression of C3 promoter activity by downregulating the transcription factor farnesoid X receptor (FXR). On the other hand, HCV NS5A protein strongly downregulated C3 promoter activity at the basal level or in the presence of interleukin-1β (IL-1β) as an inducer. In addition, the expression of the transcription factor CAAT/enhancer binding protein beta (C/EBP-β), which binds to the IL-1/IL-6 response element in the C3 promoter, was inhibited in liver biopsy specimens. Furthermore, expression of C/EBP-β was reduced in hepatocytes infected with cell culture-grown HCV, as well as in hepatocytes transfected with the NS5A genomic region of HCV. Together, these results underscore the role of HCV NS5A protein in impairing innate immune function.

Structural and Functional Analysis of the BovineMx1Promoter

The bovine Mx1 promoter region was found to contain 4 IFN-stimulated response elements (ISREs), 7 GC boxes, 2 IL-6 responsive elements, 2 NFκB-binding sites and 2 AP-1-binding sites. Among Holstein, Charolai, and Brahman breeds, 5 nucleotide substitutions were detected in the promoter region. After the Mx1 promoter region from Holstein had been constructed with pGL-basic expression vector, the transfected cells showed promoter activity after IFN induction. Several artificial deletion mutants were prepared to determine the important regulatory elements responsible for the promoter activity, and it was found that ISRE has a key function in IFN response. The proximal ISRE1 showed potential induction by IFN. Furthermore, the proximal GC boxes were found to be essential for IFN response in the bovine Mx1 promoter with the deletion mutants. In this case, the 2 GC boxes exhibited a synergistic activation in the IFN response. Mithramycin A, an agent that inhibits gene expression selectively by coating GC boxes, was used, and Mx mRNA expression in MDBK cells was suppressed by this chemical. Therefore, GC boxes were also shown to be essential for IFN response in the bovine Mx1 gene.

Identification and characterization of the prepro-vasoactive intestinal peptide gene from the teleost Paralichthys olivaceus

The gene encoding the prepro form of a vasoactive intestinal peptide (VIP) was cloned, and its structural organization and expression profiles were determined in the teleost Paralichthys olivaceus. The prepro-VIP encodes both a VIP and a peptide-histidine-isoleucine (PHI). The VIP gene comprises six exons with two distinct peptides encoded on separate exons, i.e., PHI on exon III and VIP on exon IV. Several elements involved in cytokine-mediated activation are highly conserved in an 806-bp segment of the 5′-flanking region: cAMP responsive elements (CREs), binding sites for nuclear factor IL-6 (NF-IL-6), activating protein-1 (AP-1), stimulating protein-1 (Sp-1), two IL-6 responsive element binding proteins (IL-6 RE-BPs), and signal transducers and activators of transcription (STAT). The mRNA transcripts in normally conditioned fish are expressed in the brain, intestine, stomach, pyloric ceca, spleen, and heart, but not in the muscle, liver, head or trunk kidneys, and gill. However, prepro-VIP mRNA expression in the spleen and head kidney is significantly up- and down-regulated when exposed to an artificial bacterial challenge by Edwardsiella tarda. These results suggest that the typical features of neuropeptide VIPs are evolutionarily conserved from non-mammalian vertebrates to mammals and that the flounder VIP plays an important role in the immune system, especially inflammatory processes.

IL-6 downregulates transcription of NTPDase2 via specific promoter elements

Bile ductular proliferation is markedly upregulated in biliary fibrosis and cirrhosis. However, the mechanisms regulating this upregulation in bile ductular proliferation have not been defined. Recently, we demonstrated that expression of the ectonucleotidase nucleoside triphosphate diphosphohydrolase-2 (NTPDase2/Entpd2) by portal fibroblasts (PF) is a critical regulator of bile ductular proliferation. Since interleukin 6 (IL-6) is markedly upregulated in biliary cirrhosis, our aims were to determine the role and mechanism of IL-6 in the regulation of NTPDase2 by PF. We found that IL-6 downregulated NTPDase2 protein expression in a concentration-dependent and time-dependent fashion but did not alter PF alpha-smooth muscle actin expression. IL-6 markedly downregulated NTPDase2 mRNA expression. Expression of the IL-6 receptor gp130 but not the IL-6 receptor gp80 was detected in PF. Two transcription start sites were identified in rat Entpd2 by the method of RNA ligase-mediated rapid amplification of 5' cDNA ends. The minimal promoter construct, but not shorter constructs, was downregulated by IL-6. Three putative IL-6 response elements were identified in silico and mutated. Mutation of all three response elements, but not fewer elements, completely abolished the IL-6 response. Thus IL-6 transcriptionally downregulates NTPDase2 expression by PF via actions at specific promoter elements independently of myofibroblastic differentiation. This effect may represent a novel signaling pathway by which bile ductular proliferation is dysregulated in biliary cirrhosis and thus provides a potential therapeutic approach for the regulation of bile ductular growth.

Interleukin-6 Receptor-Mediated Activation of Signal Transducer and Activator of Transcription-3 (STAT3) Promotes Choroidal Neovascularization

Interleukin (IL)-6, a potent proinflammatory cytokine, is suggested to be a risk factor for choroidal neovascularization (CNV) because of its increased levels in the serum of patients with age-related macular degeneration; however, the role of IL-6 in CNV has not been defined. The present study reveals the critical contribution of IL-6 signaling and its downstream STAT3 pathway to the murine model of laser-induced CNV. CNV induction by laser treatment stimulated IL-6 expression in the retinal pigment epithelium-choroid complex, and antibody-based blockade of IL-6 receptor or genetic ablation of IL-6 led to significant suppression of CNV. CNV generation was accompanied by STAT3 activation in choroidal endothelial cells and macrophages, and IL-6 receptor blockade resulted in selectively inhibited phosphorylation of STAT3 but not extracellular signal-regulated kinase 1/2. Consistently, pharmacological blockade of STAT3 pathway also suppressed CNV. In addition, IL-6 receptor neutralization led to significant inhibition of the in vivo and in vitro expression of inflammation-related molecules including monocyte chemotactic protein, intercellular adhesion molecule-1, and vascular endothelial growth factor, and of macrophage infiltration into CNV. These results indicate the significant involvement of IL-6 receptor-mediated activation of STAT3 inflammatory pathway in CNV generation, suggesting the possibility of IL-6 receptor blockade as a therapeutic strategy to suppress CNV associated with age-related macular degeneration.

HNF-1α plays an important role in IL-6-induced expression of the human angiotensinogen gene

Angiotensinogen (AGT) is the precursor of one of the most important vasoactive hormone angiotensin II and this gene locus is associated with human essential hypertension. AGT is an acute phase protein and its gene expression is regulated by IL-6. Previous studies have identified three potential STAT-3 binding sites (APREs) located between -160 and -280 of the hAGT gene promoter but only APRE-1 (located between -271 and -279) was shown to be a bonafide enhancer for IL-6-induced promoter activity. We show here that APRE-2, located between -236 and -247, is indeed an HNF-1alpha-binding site and plays an important role in basal and IL-6 induced promoter activity of this gene. Our chromatin immunoprecipitation (ChIP) assay shows that HNF-1alpha binds to this region of the hAGT gene promoter and its recruitment is increased in the presence of IL-6 in Hep3B cells. We also show that the promoter activity of a deletion construct containing only 223 bp of the hAGT gene promoter (that contains only APRE-3) is increased after IL-6 treatment. Our ChIP assay shows that IL-6 treatment recruits STAT-3 to APRE-3 and suggests that this is also an IL6 responsive element. We have previously shown that GR binds to the proximal promoter of the hAGT gene. Since GR physically interacts with STAT-3, we propose that transcription factors GR, STAT-3, and HNF-1alpha that bind to the nucleotide sequence located between -160 and -280 of the hAGT gene promoter are responsible for IL-6 induced promoter activity of this gene.

Interleukin-6 Transcriptionally Regulates Prohibitin Expression in Intestinal Epithelial Cells

Prohibitin (PHB) is a highly conserved protein that has multiple functions in the cell. We recently demonstrated that PHB plays an important role in combating oxidative stress and its expression is down-regulated in human and animal models of inflammatory bowel disease. Little is known regarding the regulation of PHB expression in intestine or other tissues. In this study we examined the regulation of PHB expression in intestinal epithelial cells using the model cell line Caco2-BBE. We successfully cloned the 1192-bp human PHB promoter region and identified the transcription start site 1594 bp upstream from the translation start site due to an intervening intron. We show that the acute phase cytokine interleukin-6 (IL-6) increases PHB protein and mRNA abundance and induces PHB promoter activation. The IL-6 response element site in the PHB promoter is required for maximal basal promoter activity and responsiveness to IL-6. IL-6 also increases binding of nuclear proteins to the IL-6 response element in the PHB promoter that are supershifted by a STAT3 antibody. Both basal promoter activity and IL-6 responsiveness are attenuated by signal transducer and activator of transcription 3 short interference RNA, suggesting that signal transducer and activator of transcription 3 mediates PHB activity by IL-6. Confirming these in vitro results, IL-6(-/-) mice exhibit reduced PHB expression in the colon compared with wild-type mice. These results suggest that IL-6 modulates PHB expression in cultured intestinal epithelial cells and in the intestine in vivo.

Interleukin‐6 transcriptionally regulates prohibitin expression in intestinal epithelial cells

Prohibitin (PHB) is a highly conserved protein and has pleiotropic functions in the cell. We recently demonstrated that PHB expression is decreased during inflammatory bowel disease (IBD) and that PHB protects epithelial cells from oxidant-induced barrier dysfunction. Loss of barrier function is thought to be an inciting event that underlies inflammation in IBD. Therefore, PHB may play an important role in the pathogenesis of intestinal inflammation by acting as a potent antioxidant. Little is known regarding the regulation of PHB expression in intestine or other tissues. The objective of this study was to examine the regulation of PHB expression in intestinal epithelial cells using the model cell line Caco2-BBE. We cloned the 1192 bp human PHB promoter region and identified the transcription start site 1594 bp upstream from the translation start site due to an intervening intron. The acute phase cytokine interleukin-6 (IL-6) increases PHB protein and mRNA abundance and PHB promoter activation. The IL-6 response element in the PHB promoter is required for maximal basal promoter activity and responsiveness to IL-6. IL-6 increases binding of nuclear proteins to the IL-6 response element in the PHB promoter. Basal promoter activity and IL-6 responsiveness are attenuated by SOCS-3 overexpression. Confirming these in vitro results, IL-6−/− mice exhibit reduced PHB expression in the colon compared to WT mice. These results suggest that IL-6 modulates PHB expression in cultured intestinal epithelial cells and in the intestine in vivo, an important insight given that PHB levels are altered in IBD.

Cloning and functional characterization of 5′-upstream region of metallothionein-I gene from crucian carp (Carassius cuvieri)

Metallothioneins are low molecular weight, cysteine-rich, metal-binding proteins, which can be induced by heavy metal ions, cytokines, stress, and hormones. To investigate the roles of the main cis-acting elements involved in the inducible expression of metallothionein gene in fish, the 5′-upstream region of crucian carp (Carassius cuvieri) metallothionein-I gene had been cloned and analyzed after our previous work on metallothionein-II. In its upstream region, several putative cis-acting elements, including nine metal regulatory elements (MREs), one antioxidant response element, one E-box, and three interleukin-6 responsive elements, etc. were found. The nine metal regulatory elements are confined in less than 1000bp from ATG start codon and organized into two clusters with different roles to the induction of the metallothionein-I expression. Deletion mutant assays demonstrated that both the distal and proximal clusters of metal regulatory elements contributed to the basal expression of the metallothionein-I, but only the proximal cluster was the chief contributor to the metal fold induction. In transient luciferase reporter assays, Zn2+ and Cd2+ served as much stronger inducers than Cu2+ to the metallothionein-I expression. H2O2 also could activate the metallothionein-I promoter about two-fold, which was mediated by the antioxidant response element (TGACAACGC, −437/−445). In conclusion, our studies demonstrate the roles of metal regulatory element and antioxidant response element in the induction of crucian carp metallothionein-I gene, and provide the regulatory mechanism for the use of fish metallothionein as a biomarker for monitoring of metal contamination in waters.

Characterization of IL-6 Responsive Elements in Hepcidin Promoters at the Molecular Level: Lessons from the Two Closely Related Promoter Sequences, Murine Hepc1 and Hepc2.

Hepcidin is an antimicrobial peptide that plays an important role in regulation of iron metabolism. There is one hepcidin peptide in humans and two hepcidin peptides in mice, hepcidin 1 and hepcidin 2, the murine mature peptides sharing 68% homology at the amino-acid level. The promoter region of these two genes is highly conserved (92% homology of 500 bp promoter sequence from the start of translation). However, regulation of transcription of these two genes is different. Human hepcidin and murine hepcidin 1 mRNA levels become elevated in response to iron excess and inflammation, while murine hepcidin 2 responds only to iron. Wrighting and Andrews (Blood Ahead of Print July 2006) demonstrated that the inflammatory pathway is mediated by STAT3 and that the binding site includes nucleotides −145 to −143 from start of translation in the human hepcidin promoter. Moreover, mutation of these nucleotides led to loss of IL-6 responsiveness. Alignment of the nucleotide sequences of hepcidin promoters revealed that there are two major differences between the murine hepcidin 2 (mHepc2) and the human hepcidin (hHEPC) and the murine hepcidin 1 (mHepc1) sequence in the proximal 140 bp promoter region. There is a substitution of T for A at −136 in the STAT3/AP1 site and an inversion of ApG to GpA at position −127–8 in the AP1 site. We cloned mHepc1 and mHepc2 promoters into a pGL3 basic luciferase reporter vector and mutated the two different sites in order to determine if creating the mHepc2 sequence in the mHepc1 promoter eliminated IL-6 responsiveness and if creating the mHepc1 sequence in the mHepc2 promoter restored IL-6 responsiveness of that promoter. We also created the equivalent A→T mutation in the STAT3/AP1 site in the hHEPC construct and the TpTpC→GpGpA mutation at −143–5, the same mutant as made by Wrighting and Andrews. Plasmids were transfected into human hepatoma HepG2 cells and human kidney HEK293T cells. As expected, the wildtype hHEPC reporter construct was induced 10 fold or more by IL-6. The T→A and TpTpC→GpGpA hHEPC mutants both displayed significantly reduced, but not absent, IL-6 responsiveness. The basal level of the native mHepc1-driven reporter was at least 10-fold higher than that of the native mHepc2. Furthermore, the mHepc1 promoter was similarly responsive to IL-6 as the hHEPC while the response of mHepc2 promoter was negligible. Interestingly, although the mHepc1 promoter-driven reporter manifests baseline expression even in cells of non-hepatic origin (kidney HEK293T cells), the ability to respond to IL-6 is restricted to cells of hepatic origin. We observed a significantly lower basal level of luciferase reporter activity in the mHepc1 promoter mutated to mHepc2 (A→ T and GpA→ApG), however, it was still IL-6 responsive. In contrast, complementary mutagenesis of mHepc2 to mHepc1 (T→ A and ApG→GpA) increased the basal level of reporter expression and significantly increased the mHepc2 promoter responsiveness to IL-6 although not to the extent seen with mHepc1 promoter. We conclude that the promoter region at nt −126 to −145 from the start of translation is important for the basal level of expression of hepcidin and is important for IL-6 responsiveness in the hHEPC promoter but there are probably additional regions responsible for IL-6 responsiveness in the mHepc1 promoter.

Herbal remedy magnolol suppresses IL‐6‐induced STAT3 activation and gene expression in endothelial cells

Magnolol (Mag), an active constituent isolated from the Chinese herb Hou p'u (Magnolia officinalis) has long been used to suppress inflammatory processes. Chronic inflammation is well known to be involved in vascular injuries such as atherosclerosis in which interleukin (IL)-6 may participate. Signal transducer and activator of transcription protein 3 (STAT3), a transcription factor involved in inflammation and the cell cycle, is activated by IL-6. In this study, we evaluated whether Mag can serve as an anti-inflammatory agent during endothelial injuries. The effects of Mag on IL-6-induced STAT3 activation and downstream target gene induction in endothelial cells (ECs) were examined. Pretreatment of ECs with Mag dose dependently inhibited IL-6-induced Tyr705 and Ser727 phosphorylation in STAT3 without affecting the phosphorylation of JAK1, JAK2, and ERK1/2. Mag pretreatment of these ECs dose dependently suppressed IL-6-induced promoter activity of intracellular cell adhesion molecule (ICAM)-1 that contains functional IL-6 response elements (IREs). An electrophoretic mobility shift assay (EMSA) revealed that Mag treatment significantly reduced STAT3 binding to the IRE region. Consistently, Mag treatment markedly inhibited ICAM-1 expression on the endothelial surface. As a result, reduced monocyte adhesion to IL-6-activated ECs was observed. Furthermore, Mag suppressed IL-6-induced promoter activity of cyclin D1 and monocyte chemotactic protein (MCP)-1 for which STAT3 activation plays a role. In conclusion, our results indicate that Mag inhibits IL-6-induced STAT3 activation and subsequently results in the suppression of downstream target gene expression in ECs. These results provide a therapeutic basis for the development of Mag as an anti-inflammatory agent for vascular disorders including atherosclerosis.

Interleukin‐6‐induced plasminogen gene expression in murine hepatocytes is mediated by transcription factor CCAAT/enhancer binding protein β (C/EBPβ)

An emerging area of research has demonstrated that plasminogen functions in the acute-phase response to tissue injury, neoplastic growth or infection. We have previously shown that the acute-phase mediator, interleukin (IL)-6, increases circulating plasminogen levels via upregulation of plasminogen promoter activity. We also identified a putative IL-6 responsive element (nt -791 to -783; IL6-RE) in the plasminogen gene that is required for maximal stimulation of promoter activity by IL-6. For the present study, we investigated the transcription factors and signaling pathway mediating the response of the plasminogen gene to IL-6. In electrophoretic mobility shift assays (EMSAs), a radiolabeled oligonucleotide IL6-RE probe formed specific complexes with nuclear proteins from untreated hepatocytic cells. The extent of complex formation was markedly increased using nuclear proteins from IL-6-treated cells. Complex formation was abolished by an oligonucleotide with the consensus CCAAT/enhancer binding protein (C/EBP) sequence. Furthermore, complexes were supershifted by antibodies to C/EBPbeta. Treatment of Hepa 1-6 cells with the mitogen-activated protein kinase (MAPK) inhibitor, PD-98059, inhibited IL-6-stimulated plasminogen promoter activity. These results suggest that transcription factor C/EBPbeta and the MAPK pathway play key roles in the response of the plasminogen gene to IL-6, thus elucidating a major mechanism by which the plasminogen system is upregulated to perform its crucial functions in the acute-phase response.

Molecular cloning, expression and chromosomal localization of a novel human REG family gene, REG III

Regenerating gene ( Reg), first isolated from a regenerating islet cDNA library [J. Biol. Chem. 263 (1988) 2111], encodes a secretory protein with a growth stimulating effect on pancreatic β cells that ameliorates the diabetes of 90% depancreatized rats [Proc. Natl. Acad. Sci. USA 91 (1994) 3589] and non-obese diabetic mice [Diabetes 51(Suppl. 3) (2002) S478]. Reg and Reg-related genes have been revealed to constitute a multigene family, the Reg family, which consists of four subtypes (types I, II, III, IV) based on the primary structures of the encoded proteins of the genes [Diabetes 51(Suppl. 3) (2002) S462]. Plural type III Reg genes were found in mouse and rat. On the other hand, only one type III REG gene, HIP/PAP (gene expressed in hepatocellular carcinoma-intestine-pancreas/gene encoding pancreatitis-associated protein), was found in human. In the present study, we found a novel human type III REG gene, REG III. This gene is divided into six exons spanning about 3 kilobase pairs (kb), and encodes a 175 amino acid (aa) protein with 85% homology with HIP/PAP. REG III was expressed predominantly in pancreas and testis, but not in small intestine, whereas HIP/PAP was expressed strongly in pancreas and small intestine. IL-6 responsive elements existed in the 5′-upstream region of the human REG III gene indicating that the human REG III gene might be induced during acute pancreatitis. All the human REG family genes identified so far ( REG Iα, REG Iβ, HIP/PAP, REG III and REG IV) have a common gene structure with 6 exons and 5 introns, and encode homologous 158–175-aa secretory proteins. By database searching and PCR analysis using a yeast artificial chromosome clone, the human REG family genes on chromosome 2, except for REG IV on chromosome 1, were mapped to a contiguous 140 kb region of the human chromosome 2p12. The gene order from centromere to telomere was 5′ HIP/PAP 3′-5′ RS 3′-3′ REG Iα 5′-5′ REG Iβ 3′-3′ REG III 5′. These results suggest that the human REG gene family is constituted from an ancestor gene by gene duplication and forms a gene cluster on the region.

Chylomicron-bound LPS inhibits activation of the acute phase response in vitro and in vivo

The host response to infection involves the release of multiple pro-inflammatory cytokines. These soluble mediators (e.g., TNF-α, IL-6) not only promote an acute inflammatory reaction, but also activate a dramatic change in hepatic gene expression termed the acute phase response (APR). This cytokine-mediated alteration in hepatocellular function includes changes in the activity of CCAAT/enhancer binding protein (C/EBP), an IL-6 response element that regulates many APR genes. We have previously shown the CM-bound LPS (CM-LPS) modulates the hepatocellular response to infection by inhibiting cytokine-induced NF-kB activity and NO production. Thus, we hypothesize that CM-bound LPS can similarly inhibit the IL-6-mediated activation of the APR in vitro and in vivo. For in vitro studies, rat primary hepatocytes were pretreated with or without CM-LPS and stimulated with 500 units/ml of IL-6. To enhance the IL-6 response, 1 μM dexamethasone was also added. Hepatic nuclear C/EBP was measured at 15 min and 24h after stimulation. For in vivo studies, rats were injected with CM-LPS (235 mg/kg, pre-treated) v. saline (control), recovered for 16h and then injected with IL-6 (2.6 × 105 U/kg) to induce an APR. Hepatic nuclear C/EBP and plasma LBP levels were measured at 4h and 24h after IL-6 stimulation. Pretreatment with CM-LPS markedly inhibited hepatic C/EBP activity at 15 min and 24h in vitro and at both 4 and 24h in vivo (data not shown) as compared to controls (Fig. 1). In summary, pretreatment with CM-LPS significantly inhibited activation of the APR in vitro and in vivo as measured by C/EBP activity. These data add to a growing body of evidence implicating lipoproteins as important regulators of the host immune response to bacterial infection.

Functional Analysis of Interleukin 6 Response Elements (IL-6REs) on the Human γ-Fibrinogen Promoter: BINDING OF HEPATIC Stat3 CORRELATES NEGATIVELY WITH TRANSACTIVATION POTENTIAL OF TYPE II IL-6REs

Several families of transcription factors play important roles in modulating liver-specific gene expression during an acute phase response (APR). Stat3/APR factor is the main transactivator of gene expression by the interleukin (IL)-6 family of cytokines signaling through gp130. During an APR, fibrinogen (FBG) genes are coordinately up-regulated by IL-6 and glucocorticoids. Except for rat gammaFBG, attempts to demonstrate direct binding of IL-6-activated Stat3 to FBG CTGGGAA promoter elements have not been successful. Herein we show the presence of three functional type II IL-6 response elements (IL-6REs) on the human gammaFBG promoter and that the magnitude of Stat3 binding to these elements correlates negatively with their functional activity in reporter gene assays. Stat3-specific binding to gammaFBG IL-6REs was confirmed by cross-competition with alpha2-macroglobulin IL-6RE and specific interactions with anti-Stat3 in electrophoretic mobility shift assays. All type II IL-6REs contributed to full promoter activity; however, transactivation from Site II at -306 to -301 was strongest. In contrast to a previous report, IL-6 failed to induce activation of serum amyloid A-activating factor-1/c-Myc-associated zinc finger protein (SAF-1/MAZ), and mutation of the SAF-1RE had little effect on IL-6 induction of gammaFBG promoter activity. In the absence of a functional glucocorticoid receptor response element, dexamethasone potentiated IL-6-induced gammaFBG promoter activity 2-fold, requiring promoter-proximal Site I and Site II; the promoter-distal Site III had no effect on dexamethasone potentiation of IL-6-induced promoter activity. Notably the propensity for Stat3 binding to human gammaFBG IL-6REs was low compared with Stat3 binding to the alpha2-macroglobulin IL-6RE. Together these data suggest that Stat3 transactivation via IL-6REs on FBG promoters likely involves participation of additional transcription factors and/or coactivators to achieve optimal coordinated up-regulation during an APR.