Interleukin-6 Response Element Research Articles

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.

Transcriptional regulation of myeloid differentiation primary response (MyD) genes during myeloid differentiation is mediated by nuclear factor Y

AbstractTo understand the molecular mechanism by which interleukin-6 (IL-6) regulates myeloid differentiation primary response (MyD) genes at the onset of M1 myeloid differentiation, we used JunB as a representative MyD gene to isolate and characterize IL-6 responsive elements. An IL-6 responsive element was localized between −65 and −52 of the JunB promoter (−65/−52 IL-6RE). By using antibody and oligonucleotide competition assays in electrophoretic mobility shift assay experiments, we have shown that the heterotrimeric transcription nuclear factor Y (NF-Y) complex binds to this element. A dominant-negative form of NF-YA, ectopically expressed in M1 cells, blocked NF-Y binding to the −65/−52 IL-6RE and reduced induction of JunB by IL-6. Furthermore, inhibition of NF-Y binding also reduced MyD gene induction by IL-6 and dampened the IL-6–induced M1 differentiation program. These findings are consistent with the observation that most MyD genes contain intact NF-Y binding motifs in their promoter regions. In contrast to M1 cells, during myeloid differentiation of bone marrow (BM), there was induction of NF-Y binding to the −65/−52 IL-6RE. This induced binding can be attributed to the observed induction of NF-YA protein expression and may reflect the molecular mechanism that couples proliferation to terminal differentiation of normal myeloblasts. Similar to M1 cells, blocking NF-Y binding in BM resulted in a reduction in mature macrophages. It can be concluded that NF-Y plays a role in the transcriptional regulation of MyD genes and is required for optimum myeloid differentiation.

Identification of an IL-6 response element in the human LCAT promoter

LCAT is a key enzyme of reverse cholesterol transport that is essential to maintain HDL-mediated lipid transport and cholesterol homeostasis. Alterations in LCAT expression have a profound effect on plasma HDL cholesterol concentrations. Previously LCAT mRNA and activity were shown to be regulated by several inflammatory cytokines, including the pleiotrophic cytokine interleukin-6 (IL-6). A series of full-length and sequential deletion LCAT promoter constructs were used to determine whether inflammatory stimuli affect LCAT transcription and to further identify functional, cytokine-responsive promoter regions that mediate this response. Using transfected HepG2 cells, results indicate that treatment with IL-6 induced a 2.5-fold activation of full-length LCAT promoter activity. A minimal (-1514 bp to -1508 bp) IL-6 response element with high sequence homology to the signal transducer and activator of transcription (STAT) family member, STAT3, was mapped within the distal promoter and shown to be sufficient to mediate the IL-6 response. Further, overexpression of STAT3 significantly enhanced the effect of IL-6 on LCAT promoter activity. These data suggest that the IL-6 responsive transcription factor, STAT3, contributes to LCAT transcriptional regulation. The elucidation of distinct biochemical signaling pathways associated with inflammation may provide new insight into transcriptional regulation of genes involved in lipid metabolism.

A Reg family protein is overexpressed in islets from a patient with new-onset type 1 diabetes and acts as T-cell autoantigen in NOD mice.

Genes overexpressed in pancreatic islets of patients with new-onset type 1 diabetes are potential candidates for novel disease-related autoantigens. RT-PCR-based subtractive hybridization was used on islets from a patient who died at the onset of type 1 diabetes, and it identified a type 1 diabetes-related cDNA encoding hepatocarcinoma-intestine-pancreas/pancreatic-associated protein (HIP/PAP). This protein belongs to the family of Reg proteins implicated in islet regeneration; its gene contains a putative interleukin-6 (IL-6) response element. Islets from healthy cadaveric human donors released HIP/PAP protein into the culture medium, and this release was enhanced by the addition of IL-6. The expression pattern of mouse homologues of HIP/PAP was determined in pancreata of prediabetic and diabetic NOD mice. Both groups showed positive immunostaining for HIP/PAP in islets and ductal epithelium. To test whether HIP/PAP is a target of islet-directed autoimmunity, we measured splenic T-cell responses against HIP/PAP in NOD mice. Spontaneous proliferation was detected after 4 weeks. Lymphocytes from islet infiltrates and pancreatic lymph nodes from 7- to 10-week-old NOD mice were used to establish an HIP/PAP-specific I-A(g7)-restricted T-cell line, termed WY1, that also responded to mouse islets. WY1 cells homed to islets of NOD-SCID mice and adoptively transferred disease when coinjected with purified CD8(+) cells from diabetic NOD mice. Our conclusion was that differential cloning of Reg from islets of a type 1 diabetic patient and the response of Reg to the cytokine IL-6 suggests that HIP/PAP becomes overexpressed in human diabetic islets because of the local inflammatory response. HIP/PAP acts as a T-cell autoantigen in NOD mice. Therefore, autoimmunity to HIP/PAP might create a vicious cycle, accelerating the immune process leading to diabetes.

Transcriptional control mechanism of fibrinogen gene expression.

Although fibrinogen genes are expressed constitutively in hepatocytes, their transcription can be greatly increased during inflammatory stress. Extensive studies have focused on the cytokine mediated transcriptional regulation of fibrinogen genes. It is clear that interleukin-6 (IL-6) and its family of cytokines are the major inducers of fibrinogen gene expression. Functional analyses of all three fibrinogen promoters for human and rat all demonstrate that the conserved CTGGGAA motifs within the proximal promoter of each fibrinogen gene are the IL-6 responsive elements. Exploration of the rat gamma fibrinogen gene demonstrated that the IL-6 activated transcription factor, STAT3, binds to the CTGGGAA motif and is required for the IL-6 mediated upregulation of this gene. IL-6 mediated fibrinogen production can be significantly elevated by glucocorticoid treatment. The synergistic effect of glucocorticoids and IL-6 relies on the functional interaction between STAT3 and glucocorticoid receptor. In addition to the upregulation signals for fibrinogen gene expression during inflammatory stress, other signaling also downregulates the expression of fibrinogen genes. For example, the proinflammatory cytokine IL-1 beta exerts inhibitory function on IL-6 mediated fibrinogen gene expression. Given the fact that elevated levels of fibrinogen in blood correlate with increased risk for cardiovascular disease, there is strong motivation to explore the molecular mechanisms that control fibrinogen expression, especially those signals that may downmodulate expression and thus provide novel approaches to controlling fibrinogen levels.

C-Jun AND c-Fos COOPERATE WITH STAT3 IN IL-6-INDUCED TRANSACTIVATION OF THE IL-6 RESPONSE ELEMENT (IRE)

Transcriptional activation of eukaryotic genes often requires the cooperative action of many proteins. The interleukin 6 (IL-6) response element (IRE) is activated by signal transducer and activator of transcription 3 (STAT3), and stimulation with IL-6 leads to STAT3 tyr705 phosphorylation, dimerization, translocation to the nucleus and transactivation of target gene promoters containing IREs. Here, we report that IL-6 and 12-O-tetradecanoylphorbol-13-acetate (TPA) synergistically transactivate the IRE in HepG2 cells, which is coupled to a strong upregulation of c-Jun and c-Fos expression by TPA via the mitogen-activated protein kinase (MAPK) pathway. Overexpression of c-Jun and c-Fos strongly enhanced STAT3-driven IRE transactivation as well as transactivation of the human intercellular adhesion molecule (ICAM)-1 promoter. In contrast, c-Jun mutants lacking the transactivation domain, the DNA-binding domain, or mutants in which the serine residues 63 and 73 were replaced by alanine, did not cooperate with STAT3. In immunoprecipitation experiments, a direct association of STAT3 with c-Jun and c-Fos was observed in response to IL-6. Furthermore, c-Jun/STAT3 and c-Fos/STAT3 complexes were detected on IRE probes in electrophoretic mobility shift assay (EMSA) experiments, but did not bind nor transactivate the TPA response element (TRE). These results demonstrate that activator protein-1 (AP-1) transcription factors can cooperate with STAT3 in IRE transactivation in the absence of direct AP-1 DNA binding.

Interleukin-6-induced Tethering of STAT3 to the LAP/C/EBPβ Promoter Suggests a New Mechanism of Transcriptional Regulation by STAT3

LAP/C/EBPbeta is a member of the C/EBP family of transcription factors and contributes to the regulation of the acute phase response in hepatocytes. Here we show that IL-6 controls LAP/C/EBPbeta gene transcription and identify an IL-6 responsive element in the LAP/C/EBPbeta promoter, which contains no STAT3 DNA binding motif. However, luciferase reporter gene assays showed that STAT3 activation through the gp130 signal transducer molecule is involved in mediating IL-6-dependent LAP/C/EBPbeta transcription. Southwestern analysis indicated that IL-6 induces binding of a 68-kDa protein to the recently characterized CRE-like elements in the LAP/C/EBPbeta promoter. Transfection experiments using promoter constructs with mutated CRE-like elements revealed that these sites confer IL-6 responsiveness. Further analysis using STAT1/STAT3 chimeras identified specific domains of the protein that are required for the IL-6-dependent increase in LAP/C/EBPbeta gene transcription. Overexpression of the amino-terminal domain of STAT3 blocked the IL-6-mediated response, suggesting that the STAT3 amino terminus has an important function in IL-6-mediated transcription of the LAP/C/EBPbeta gene. These data lead to a model of how tethering STAT3 to a DNA-bound complex contributes to IL-6-dependent LAP/C/EBPbeta gene transcription. Our analysis describes a new mechanism by which STAT3 controls gene transcription and which has direct implication for the acute phase response in liver cells.

Interleukin 1β inhibits interleukin 6–mediated rat γ fibrinogen gene expression

Interleukin (IL)-1β and IL-6 are the 2 major inducers of a group of hepatic genes during acute inflammation; however, each cytokine uses different intracellular signaling molecules. In most instances, the 2 cytokines interact positively to enhance hepatic gene expression, but in one class of acute-phase reactants, which includes fibrinogen, IL-1β exerts a transient inhibitory effect over the IL-6 stimulatory signal. This study explored the effects of IL-1β/nuclear factor κB (NF-κB) and IL-6/signal transducer and activator of transcription 3 (STAT3) combinatory signaling on the transcriptional regulation of the rat γ fibrinogen gene. Northern blot and functional analyses employing luciferase reporter constructs driven by the rat γ fibrinogen promoter demonstrated that IL-1β inhibited the IL-6-mediated transcription of this gene. Exposing primary rat hepatocytes to IL-1β had no effect on IL-6-mediated STAT3 activation; instead, IL-1β-activated NF-κB associated with 2 IL-6 responsive elements (STAT3 binding site) on the rat γ fibrinogen promoter and blocked STAT3 binding to these regions. The competitive binding of NF-κB and STAT3 on the overlapping binding site provides a mechanism for the inhibition by IL-1β of the IL-6-mediated transactivation of rat γ fibrinogen.

Interleukin 1β inhibits interleukin 6–mediated rat γ fibrinogen gene expression

AbstractInterleukin (IL)-1β and IL-6 are the 2 major inducers of a group of hepatic genes during acute inflammation; however, each cytokine uses different intracellular signaling molecules. In most instances, the 2 cytokines interact positively to enhance hepatic gene expression, but in one class of acute-phase reactants, which includes fibrinogen, IL-1β exerts a transient inhibitory effect over the IL-6 stimulatory signal. This study explored the effects of IL-1β/nuclear factor κB (NF-κB) and IL-6/signal transducer and activator of transcription 3 (STAT3) combinatory signaling on the transcriptional regulation of the rat γ fibrinogen gene. Northern blot and functional analyses employing luciferase reporter constructs driven by the rat γ fibrinogen promoter demonstrated that IL-1β inhibited the IL-6-mediated transcription of this gene. Exposing primary rat hepatocytes to IL-1β had no effect on IL-6-mediated STAT3 activation; instead, IL-1β-activated NF-κB associated with 2 IL-6 responsive elements (STAT3 binding site) on the rat γ fibrinogen promoter and blocked STAT3 binding to these regions. The competitive binding of NF-κB and STAT3 on the overlapping binding site provides a mechanism for the inhibition by IL-1β of the IL-6-mediated transactivation of rat γ fibrinogen.

Tumour Necrosis Factor-α Regulation of Prostaglandin H Synthase-2 Transcription is not through Nuclear Factor-κB in Amnion-derived AV-3 Cells

Tumour necrosis factor (TNF)-α-stimulated prostaglandin (PG) E2biosynthesis by amnion-derived AV3 cells is accompanied by increased prostaglandin H synthase (PGHS)-2 mRNA expression. PGHS-1 mRNA expression is unchanged. PGHS-2 promoter–reporter constructs (−891/+9 and 5′ deletions thereof) were prepared. The regions containing concensus nuclear factor κB (NF-κB) elements (−447/−438 and −222/−213) did not enhance promoter activity. Elements associated with both basal and TNF-α-stimulated expression lie between bases −52 and −203. Site-directed mutagenesis of nuclear factor of interleukin-6 (NF-IL6) and cyclic AMP response elements (CREs) in this region reduced both basal and induced transcriptional activity of the −203/+9 construct by over 95 per cent. Electrophoretic mobility-shift assays using oligonucleotides derived from these sites demonstrated formation of specific DNA–protein complexes. Both NF-IL6 and CRE unlabelled oligonucleotides inhibited complex formation with the NF-IL6 oligonucleotide probe. Unlabelled CRE oligonucleotide also effectively inhibited formation of the complex with the CRE probe, but reduced effectiveness was observed when the NF-IL6 oligonucleotide was the competitor. Finally, unlabelled, consensus NF-κB oligonucleotide failed to compete for either probe. TNF-α treatment did not increase levels of these complexes. Thus NF-κB does not enhance basal or TNF-α-responsive PGHS-2 transcription in amnion-derived AV-3 cells. A permissive role for NF-IL6/CRE binding proteins in regulating PGHS-2 expression in these cells is indicated, but requires further clarification.

A SAF binding site in the promoter region of human gamma-fibrinogen gene functions as an IL-6 response element.

Expression of fibrinogen is highly induced during inflammation, and such abnormal expression of this protein is considered as a major cardiovascular risk factor. IL-6 is one of the main mediators of abnormal expression of fibrinogen leading to the pathogenic conditions. Transient transfection and EMSA were performed to investigate the molecular mechanism of IL-6-induced gamma-fibrinogen gene expression in hepatic cells. Using progressively deleted 5' fragments of the gamma-fibrinogen promoter coupled to chloramphenicol acetyltransferase gene, an IL-6 responsive element located between positions -273 and -259 was identified. Mutation of this element abrogates IL-6 responsiveness of the gamma-fibrinogen promoter. Interaction of this promoter with a zinc finger transcription factor, serum amyloid A activating factor (SAF)-1, was demonstrated by EMSA. Furthermore, overexpression of wild-type SAF-1 in transfected liver cells can increase transcription of the gamma-fibrinogen promoter. These data show that transcription factor SAF-1 is involved in the regulation of IL-6-mediated induction of the human gamma-fibrinogen gene in liver cells.

KGF and FGF-10 stimulate liquid secretion in human fetal lung.

During fetal life, the pulmonary epithelium secretes liquid that distends the airways and is important for normal lung growth and development. The factors regulating human fetal lung liquid secretion are poorly understood; however, recent studies in murine models show that keratinocyte growth factor (KGF, FGF-7) and fibroblast growth factor 10 (FGF-10) stimulate liquid secretion. We asked whether KGF and FGF-10 stimulate liquid secretion in human fetal lung. First trimester fetal lung explants developed dose-dependent increases in intraluminal volume in response to KGF and FGF-10. Although there were no acute changes in explant transepithelial potential difference in response to KGF (0.1-1000 ng/mL), exposure to 5-50 ng/mL KGF over 60 h depolarized transepithelial potential difference compared with controls. We used ribonuclease protection assays to quantitate the ontogeny and regulation of mRNA expression for KGF and its receptor. Both mRNA were expressed in fetal and postnatal lung. Because the promoter region of the human KGF gene contains cAMP and IL-6 response elements, we asked whether cAMP or IL-6 stimulated expression of KGF or its receptor. We have previously shown that cAMP stimulates liquid secretion in this model. Both cAMP and IL-6 significantly increased expression of KGF but not KGF receptor during a 48-h experiment. Thus, stimulation of liquid secretion in explant models by cAMP may be mediated in part by induction of KGF expression. KGF and FGF-10 may be important paracrine factors regulating liquid secretion in human fetal lung.

Interleukin-6 and Leukemia Inhibitory Factor Induction of JunB Is Regulated by Distinct Cell Type-specific Cis-acting Elements

Interleukin (IL)-6 plays an important role in a wide range of biological activities, including differentiation of murine M1 myeloid leukemic cells into mature macrophages. At the onset of M1 differentiation, a set of myeloid differentiation primary response (MyD) genes are induced, including the proto-oncogene for JunB. In order to examine the molecular nature of the mechanisms by which IL-6 activates the immediate early expression of MyD genes, JunB was used as a paradigm. A novel IL-6 response element, -65/-52 IL-6RE, to which a 100-kDa protein complex is bound, has been identified on the JunB promoter. Leukemia inhibitory factor (LIF)-induced activation of JunB in M1 cells was also mediated via the -65/-52 IL-6RE. The STAT3 and CRE-like binding sites of the JunB promoter, identified as IL-6-responsive elements in HepG2 liver cells were found, however, to play no role in JunB inducibility by IL-6 in M1 myeloid cells. Conversely, the -65/-52 IL-6RE is shown not to be necessary for JunB inducibility by IL-6 or LIF in liver cells. It appears, therefore, that immediate early activation of JunB is regulated differently in M1 myeloid cells than in HepG2 liver cells. This indicates that distinct cis-acting control elements participate in cell type-specific induction of JunB by members of the IL-6 cytokine superfamily.

KSHV-encoded viral IL-6 activates multiple human IL-6 signaling pathways

Kaposi’s sarcoma-associated herpesvirus (KSHV or HHV8) encodes a structural and functional homologue of human IL-6 called viral IL-6 (vIL-6). Expression of vIL-6 in KSHV-related lymphoproliferative disorders has been implicated in their pathogenesis. vIL-6 has been shown to mimic a number of IL-6 activities including stimulating the growth of IL-6 dependent cell lines and activating the JAK1 and STAT1/3 pathway in HepG2 cells. However, IL-6 and vIL-6 display differences in receptor usage that may give rise to underlying qualitative and quantitative differences in the signaling pathways utilized. While IL-6 has an absolute requirement for both the IL-6 Rα and the gp130 subunits, vIL-6 appears to require only gp130. In addition to JAK1 and STAT1/3 pathways, IL-6 activates multiple other pathways including the direct activation of STAT 5 by JAK1, the Ras-MAP kinase cascade and a novel H7-sensitive pathway. In this study we examined whether vIL-6 is capable of signaling via distinct IL-6 response elements (IL-6 RE) under the control of these different pathways. We show that vIL-6 activates both STAT1/3- and STAT5-dependent Type II IL-6 REs. In addition, vIL-6 induces transcriptional activation via a Type I IL-6 RE that binds C/EBP, indicative of Ras-MAP kinase pathway induction. Furthermore, vIL-6 is capable of activating the IL-6 response element in the c-jun promoter (JRE-IL-6). vIL-6 induced activation of JRE-IL-6 requires both the Ets- and Cre-like sites, suggesting that vIL-6 is capable of stimulating the same novel serine/threonine kinase mediated pathway as IL-6. These results demonstrate that vIL-6 can stimulate all of the known IL-6-induced signaling pathways. Therefore, vIL-6 could potentially contribute to KSHV-related disease progression by continued activation of IL-6-stimulated growth and anti-apoptotic pathways even when cells attempt to protect themselves from IL-6 over-stimulation by downmodulating their IL-6Rα subunits.

New Horizons in Orthopaedic Research

The report by Nakashima et al.6 in this issue of The Journal and the one by Blaine et al.3 in the October 1997 issue dealt with signal transduction mechanisms in macrophages stimulated by orthopaedic biomaterials. Although these pioneering studies represent initial findings and focus on only a narrow range of possible signaling events, they open new vistas within clinically oriented orthopaedic research. The ultimate aim of this new line of research is to delineate the fundamental cellular mechanisms of periprosthetic osteolysis induced by particulate wear debris and to establish strategies for the development of therapeutic agents to prevent, reverse, or retard this adverse outcome of joint replacement procedures. Furthermore, the study of cellular signal transduction pathways can be applied to numerous areas of orthopaedic research, including the growth and development of cartilage and bone, the pathogenesis of osteoarthritis, fracture-healing, and tumor growth, to name but a few. One of the most important advances in biomedical science in this decade is the identification of intracellular signaling events as part of cellular adaptive responses. These regulatory events are important for mammalian cells to deal with changes in their microenvironment and maintain homeostasis. Cells receive external signals by way of surface membrane receptors, and as a consequence certain amino acids (usually tyrosine, serine, or threonine residues) of the cytoplasmic tail of the receptor become phosphorylated. This initial biochemical event is amplified through a series of protein kinase phosphorylations that in the end trigger a defined set of intracellular adaptive processes, including long-term gene responses. Extracellular compounds in the form of lipids, hormones, growth factors, cytokines, chemokines, counter receptors of other cells, or other cellular mediators can bind to specific cell-surface receptors. These interactions trigger intracellular signal-transduction cascades that result in the modulation of nuclear transcription factor activity. Transcription factors are nuclear …

Crosstalk between the interleukin-6 (IL-6)-JAK-STAT and the glucocorticoid-nuclear receptor pathway: synergistic activation of IL-6 response element by IL-6 and glucocorticoid.

Cytokines and steroid hormones use different sets of signal transduction pathways, which seem to be unrelated. Interleukin-6 (IL-6) uses JAK tyrosine kinase and STAT (signal transducer and activator of transcription) transcription factor. Glucocorticoid binds glucocorticoid receptor (GR), which is a member of the steroid receptor superfamily. We have studied the crosstalk between the IL-6-JAK-STAT and glucocorticoid-nuclear receptor pathways. IL-6 and glucocorticoid synergistically activated the IL-6 response element on the rat alpha2-macroglobulin promoter (APRE)-driven luciferase gene. The exogenous expression of GR enhanced the synergism. The exogenous expression of dominant negative STAT3 completely abolished the IL-6 plus glucocorticoid-induced activation of the APRE-luciferase gene. Tyrosine phosphorylation of STAT3 stimulated by IL-6 alone was not different from that by IL-6 plus glucocorticoid. The protein level of STAT3 was also not increased by glucocorticoid stimulation. The time course of STAT3 tyrosine phosphorylation by IL-6 plus glucocorticoid was not different from that by IL-6 alone. The synergism was studied on the two other IL-6 response elements, the junB promoter (JRE-IL-6) and the interferon regulatory factor-1 (IRF-1) promoter (IRF-GAS) which could be activated by STAT3. The synergistic activation by glucocorticoid on the IL-6-activated JRE-IL-6 and the IRF-GAS-driven luciferase gene was not detected. Glucocorticoid did not change the mobility of IL-6-induced APRE-binding proteins in a gel shift assay. These results suggest that the synergism was through the GR and STAT3, and the coactivation pathway which was specific for APRE was the target of glucocorticoid.

Requirement for Cooperative Interaction of Interleukin-6 Responsive Element Type 2 and Glucocorticoid Responsive Element in the Synergistic Activation of Mouse Metallothionein-I Gene by Interleukin-6 and Glucocorticoid

Metallothionein (MT)-inducing activity of interleukin (IL)-6 depends on the presence of glucocorticoid in hepatic cells. The synergistic action of IL-6 and glucocorticoid was observed in the transcriptional activation of the mouse MT (mMT)-I gene. We found that a 281-bp promoter was sufficient for IL-6 and glucocorticoid stimulation. Our inspection of this region revealed the putative type 1 and 2 IL-6 responsive elements (REs). Functional analyses of these regions were performed using luciferase reporter constructs, and it was observed that the type 2 IL-6RE exerted the major response to the IL-6 signal. The transcriptional factor binding to type 1 IL-6RE, nuclear factor-IL-6, hardly contributed to the activation of the mMT-I promoter by IL-6 and glucocorticoid. A glucocorticoid responsive element (GRE) was also required for the synergistic activation by IL-6 and glucocorticoid. Interestingly, this synergism was not observed when the type 2 IL-6RE and the GRE were kept apart. Therefore, the synergistic activation of the mMT-I gene by IL-6 and glucocorticoid may require not only that signal transducers and activators 3 (Stat3) and the glucocorticoid receptor (GR) bind to their respective responsive elements, but also that Stat3 and the GR physically interact with one another.

A unique transcription factor for the A alpha fibrinogen gene is related to the mitochondrial single-stranded DNA binding protein P16.

Although stimulation of hepatic cells with interleukin-6 induces the expression of fibrinogen, the molecular basis for this regulation remains largely uncharacterized. A recent examination of the A alpha fibrinogen gene promoter identified a protein, termed the A alpha-core protein, that bound constitutively to the IL-6 response element [Liu, Z. & Fuller, G. M. (1995) J. Biol. Chem. 270, 7580-7586]. This current study provides further characterization of this regulatory protein. The data presented show the following: (i) The A alpha-core protein has a similar molecular weight and identical N-terminal sequence to that of the mitochondrial single-stranded DNA binding protein P16. (ii) The A alpha-core protein and P16 have similar characteristics in terms of DNA binding preference and antigenic properties. (iii) Overexpression of P16 gene in the hepatoma cell lines Hep G2 and Hep 3B enhances the IL-6-induced expression of A alpha fibrinogen. These results demonstrate that the A alpha-core protein is closely related to P16 and involved in the IL-6-regulated transcription of A alpha fibrinogen.

Transcriptional activation of the intercellular adhesion molecule 1 (CD54) gene by human T lymphotropic virus types I and II Tax is mediated through a palindromic response element.

In vitro infection of T cells with human T lymphotropic virus types I and II (HTLV-I and HTLV-II) resulted in constitutive expression of ICAM-1. Higher levels of ICAM-1 mRNA were expressed in HTLV-transformed cell lines (MT-2, MoT, C8166) when compared with uninfected T cell lines (A301). We demonstrate that this activation is conferred through a site on the ICAM-1 promoter that is activated in trans by the Tax protein of HTLV-I and HTLV-II. Enhanced promoter activity was detected when the ICAM-1 construct (-1162/+1) was transfected into HTLV-I-infected (MT-2), HTLV-II-infected (MoT, AI 1050), or an HTLV-I Tax-only-expressing (C8166) cell line as compared to the uninfected T cell line (A3.01). Cotransfection of the uninfected T cell line A3.01 with the ICAM construct along with Tax-I and Tax-II expression plasmid also resulted in increased promoter activity. Furthermore, experiments with deletion constructs of the ICAM-1 promoter region indicated that a region between -88 and -53 bp relative to the transcription start site is sufficient for Tax-inducible CAT expression. This segment includes an 11-bp palindromic segment (TTTCCGGGAAA) that has homology with the IFN-gamma and IL-6 response element. An 11-bp segment containing this regulatory region proved to be sufficient to confer Tax-I and Tax-II inducibility on a heterologous promoter (TK-CAT). Taken together these findings indicate that constitutive expression of ICAM-1 by HTLV-infected cells is influenced by the viral trans-activator protein Tax. This increased expression of ICAM-1 in response to the Tax protein may play an important role in the lymphoproliferation associated with HTLV infection.

Globin Gene Silencing in Primary Erythroid Cultures: AN INHIBITORY ROLE FOR INTERLEUKIN-6

There are numerous similarities between the erythroid and megakaryocytic lineages which suggest that commitment to either lineage occurs relatively late in hematopoiesis. Commitment toward megakaryocyte development requires obligatory silencing of erythroid-specific genes. Therefore, we investigated the effects of interleukin-6, a known inducer of thrombocyte production, on globin gene expression during erythroid differentiation. Studies in K562 cells demonstrated inhibition of gamma globin gene mRNA production and chain biosynthesis in the presence of exogenous interleukin-6 which was abrogated by anti-interleukin-6 monoclonal antibody. Similar studies in primary erythroid progenitors showed inhibition of burst-forming unit-erythroid colony formation when interleukin-6 was added late in cultures with decreased gamma and beta globin gene mRNA production. Protein binding studies demonstrated an increase in activator protein-1 binding to its consensus sequence by 24 h of interleukin-6 treatment. Inhibition of activator protein-1 gene activity had no effect on gamma gene silencing by interleukin-6. A potential interleukin-6 response element was identified in the gamma globin gene. Interleukin-6 treatment led to a rapid increase in protein binding to the target DNA sequence. These results suggest that interleukin-6 may play an important role in globin gene silencing during megakaryocytic lineage commitment.