—Interleukin-6 (IL-6) is a multifunctional cytokine expressed by angiotensin II (Ang II)-stimulated vascular smooth muscle cells (VSMCs) that functions as an autocrine growth factor. In this study, we analyze the mechanism for Ang II-inducible IL-6 expression in quiescent rat VSMCs. Stimulation with the Ang II agonist Sar 1 Ang II (100 nmol/L) induced transcriptional expression of IL-6 mRNA transcripts of 1.8 and 2.4 kb. In transient transfection assays of IL-6 promoter/luciferase reporter plasmids, Sar 1 Ang II treatment induced IL-6 transcription in a manner completely dependent on the nuclear factor-κB (NF-κB) motif. Sar 1 Ang II induced cytoplasmic-to-nuclear translocation of the NF-κB subunits Rel A and NF-κB1 with parallel changes in DNA-binding activity in a biphasic manner, which produced an early peak at 15 minutes followed by a nadir 1 to 6 hours later and a later peak at 24 hours. The early phase of NF-κB translocation was dependent on weak simultaneous proteolysis of the IκBα and β inhibitors, whereas later translocation was associated with enhanced processing of the p105 precursor into the mature 50-kDa NF-κB1 form. Pretreatment with a potent inhibitor of IκBα proteolysis, TPCK, completely blocked Sar 1 Ang IIAng II-induced NF-κB activation and induction of endogenous IL-6 gene expression, which indicated the essential role of NF-κB in mediating IL-6 expression. We conclude that Ang II is a pleiotropic regulator of the NF-κB transcription factor family and may be responsible for activating the expression of cytokine gene networks in VSMCs.

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The gene encoding angiotensinogen, the glycoprotein precursor of the potent vasopressor peptide angiotensin II, is transcriptionally activated in hepatocytes during the acute-phase response through interactions of mutually cooperative glucocorticoid receptors and proteins that bind to an acute-phase response element (APRE) 5'-AGTTGGGATTTCCCAACC-3'. The APRE binds a family of constitutive proteins (BPcs) and a cytokine inducible protein (BPi) that is indistinguishable from nuclear factor xB (NFxB). The interactions of purified proteins with the APRE were studied by in vitro binding and in vivo transcriptional trans-activation assays. BPc is a family of heat-stable DNA binding proteins, the different sized members of which are capable of forniing heterodimers. BPcs are recognized by anti-C/EBP antiserum and produce a footprint similar to bacterially expressed C/EBP on the APRE. BPi has a 4-to 5-fold greater affinity for the APRE than the BPcs, and contacts guanosine residues distinct from those contacted by the BPcs, demonstrating that these two classes of proteins contain functionally distinct DNA binding domains.

Recent studies indicate that maximal IL-8 protein expression requires activation of NF-κB as well as activation of the MAP kinases ERK, JNK, and p38. However, the precise relationship between NF-κB transactivation and MAP kinase activation remains unclear. We examined the requirements of NF-κB, ERK, JNK, and p38 for TNF-α-induced transcription from the IL-8 promoter in a human bronchial epithelial cell line. Treatment with TNF-α induced activation of all three MAP kinases. Using a combination of chemical and dominant-negative inhibitors, we found that inhibition of NF-κB, ERK, and JNK, but not p38, each decreased TNF-α-induced transcription from the IL-8 promoter. Inhibition of JNK signaling also substantially reduced TNF-α-induced NF-κB transactivation, whereas inhibition of ERK and p38 had no effect. On the other hand, ERK was required and sufficient for TNF-α-induced activation of activator protein (AP)-1 promoter sequences, which together function as a basal level enhancer. JNK activation was also required for AP-1 transactivation. Finally, inhibition of p38 attenuated IL-8 protein abundance, suggesting that p38 regulates IL-8 expression in a posttranscriptional manner. We conclude that, in human airway epithelial cells, MAP kinases may regulate IL-8 promoter activity by NF-κB-dependent (in the case of JNK) and -independent (ERK) processes, as well as by posttranscriptional mechanisms (p38).

The intravascular renin-angiotensin system is an endocrine system designed to maintain cardiovascular homeostasis in response to hypotension. Under normal conditions, angiotensinogen concentrations circulating in the plasma are rate limiting for the maximum velocity of angiotensin I formation. In the liver, the major site of circulating angiotensinogen synthesis, angiotensinogen expression is under exquisite hormonal control. We review the mechanisms by which hormones effect transcriptional control of angiotensinogen expression. Adrenal-derived glucocorticoids produce the translocation of the glucocorticoid receptor into the nucleus. It in turn binds to two glucocorticoid response elements and stimulates angiotensinogen gene transcription. Inflammation activates angiotensinogen transcription as a result of the macrophage-derived cytokines interleukin-1 and tumor necrosis factor-alpha. These cytokines change the abundance of two transcription factor families that bind a single regulatory site in the angiotensinogen promoter, the acute-phase response element. These proteins include the nuclear factor-kappaB complex and the CCAAT/enhancer binding protein family. Activation of the renin-angiotensin system, through production of angiotensin II, results in feedback stimulation of angiotensinogen synthesis (the "positive feedback loop"). We have discovered that the nuclear factor-kappaB transcription factor is regulated by angiotensin II, a finding that provides a mechanism for the transcriptional component of angiotensinogen gene synthesis in the positive feedback loop. These studies underscore the concept that induction of the angiotensinogen gene by diverse physiological stimuli is mediated through changes in the nuclear abundance of sequence-specific transcription factors. The intracellular convergence of cytokine- and angiotensin II-induced signaling pathways on the nuclear factor-kappaB transcription factor provides a point for "cross talk" between angiotensin- and cytokine-activated second messenger pathways.

The alveolar macrophage-derived peptide tumor necrosis factor-␣ (TNF␣) initiates pulmonary inflammation through its ability to stimulate interleukin-8 (IL-8) synthesis in alveolar epithelial cells through an incompletely described transcriptional mechanism. In this study, we use the technique of ligation-mediated polymerase chain reaction (LMPCR) to record changes in transcription factor occupancy of the IL-8 promoter after TNF␣ stimulation of A549 human alveolar cells. Using dimethylsulfate/LMPCR, no detectable proteins bind the TATA box in unstimulated cells. By contrast, TNF␣ rapidly induces protection of G residues at ؊79 and ؊80 coincident with endogenous IL-8 gene transcription. Using DNase I/LMPCR, we observe inducible protection of nucleotides ؊60 to ؊99 (the TNF response element) and nucleotides ؊3 to ؊32 (containing the TATA box). Surprisingly, extensive TATA box protection is only seen after TNF␣ stimulation. Using a two-step microaffinity isolation/Western immunoblot DNA binding assay, we observe that the NF-B subunits Rel A, NF-B1, and c-Rel inducibly bind the TNF response element; these proteins undergo rapid TNF␣-inducible increases in nuclear abundance as a consequence of IB␣ proteolysis. Furthermore, the peptide aldehyde N-acetyl-Leu-Leu-norleucinal, an agent that blocks both IB␣ proteolysis and NF-B subunit translocation, abrogates recombinant human TNF␣-inducible IL-8 gene transcription. These studies demonstrate that IL-8 is activated by a promoter recruitment mechanism in alveolar epithelial cells, where NF-B subunit translocation is required for (and coincident with) binding of the constitutively active TATA box-binding proteins.The respiratory epithelium contributes to normal pulmonary function in its ability to clear inhaled particulates through mucociliary action, ensure alveolar patency through surfactant secretion, and facilitate bacterial opsonization through secretory immunoglobulin production. A large body of evidence now supports an additional role for the airway epithelial cell to amplify cytokine signals from pathogen-activated alveolar macrophages into secretion of chemokines, arachidonic acid metabolites, and phospholipid-molecules that recruit additional inflammatory cells into the airway mucosa (1, 2). Of the potent alveolar macrophage-derived cytokines studied, the secretion of tumor necrosis factor ␣ (TNF␣), 1 in particular, has been implicated in the pathophysiology of neutrophilic-infiltrating disorders including acute lung injury from sepsis, silica-induced pulmonary fibrosis, allograph rejection, and acute respiratory tract infection (3-6).The peptide hormone TNF␣ activates signaling cascades by inducing trimerization of the airway epithelial cell-expressed 55-kDa TNF receptor type I (TNFR1). Liganded trimeric TNFR1, in turn, recruits TNF receptor-associated proteins (TRADD, FADD, TRAF2, and others) to its intracytoplasmic domain to generate intracellular signaling cascades via second messengers including ceramide, 1,2-diacylglycerol, and arachidonic acid metabolites (s...

Angiotensinogen is the glycoprotein precursor of angiotensin II, an octapeptide hormone important for the regulation of blood pressure and volume homeostasis. The gene encoding angiotensinogen is expressed in liver and several other tissues, providing a model gene for understanding the role of cis-acting DNA control elements and trans-acting factors in tissue-type specific gene expression. To identify DNA control elements in the rat angiotensinogen gene we prepared an array of fusion genes consisting of either 5' or 3'-deleted sequences of the 5'-flanking region of the gene linked to a firefly luciferase reporter gene and analyzed the relative cellular specificity of expression of these genes after their introduction into hepato-carcinoma cells (Hep G2) that do express and placental cells (JEG-3) that do not express the endogenous angiotensinogen gene. Six transcriptionally active elements were found within 688 base pairs of 5'-flanking DNA. The interactions of DNA binding proteins with these elements was demonstrated by their specific protection to digestion with DNase I in the presence of liver cell extracts. The orientation and spatial requirements for transcription of two of the elements were analyzed further by the construction and expression of synthetic oligonucleotide cassettes incorporating the sequences of these elements when linked to a homologous (angiotensinogen) or a heterologous Simian virus 40 promoter and enhancer. One element located between 60 and 108 base pairs from the start of gene transcription functioned either as a silencer or an enhancer of transcription (SOAP box element), depending upon the distance from the angiotensinogen and viral gene promoters. Moreover, the SOAP box element demonstrated enhancer activity in JEG-3 cells when linked to the Simian virus 40 early promoter. An oligonucleotide mutation of the SOAP box element interfered with protein binding in a gel mobility shift assay and this mutant was transcriptionally inactive in both homologous and heterologous promoters. These observations indicate that expression of the angiotensinogen gene in liver cells is coordinately regulated by multiple cis-acting elements that interact with DNA binding proteins.

The proinflammatory cytokine, tumor necrosis factor ␣ (TNF␣), is a potent activator of angiotensinogen gene transcription in hepatocytes by activation of latent nuclear factor-B (NF-B) DNA binding activity. In this study, we examine the kinetics of TNF␣-activated translocation of the 65-kDa (Rel A) and 50-kDa (NF-B1) NF-B subunits mediated by inhibitor (IB) proteolysis in HepG2 hepatoblastoma cells. HepG2 cells express the IB members IB␣, IB␤, and IB␥. In response to TNF␣, Rel A⅐NF-B1 translocation and DNA binding activity follows a biphasic profile, with an "early" induction (15-30 min), followed by a nadir to control levels at 60 min, and a "late" induction (>120 min). The early phase of Rel A⅐NF-B1 translocation depends on simultaneous proteolysis of both IB␣ and IB␤ isoforms; IB␥ is inert to TNF␣ treatment. The 60-min nadir is due to a rapid IB␣ resynthesis that reassociates with Rel A and completely inhibits its DNA binding activity; the 60-min nadir is not observed when IB␣ resynthesis is prevented by cycloheximide treatment. By contrast, selective inhibition of IB␤ proteolysis by pretreatment of HepG2 cells with the peptide aldehyde N-acetyl-Leu-Leu-norleucinal completely blocks the late phase of Rel A⅐NF-B1 translocation. These studies indicate the presence of inducible and constitutive cytoplasmic NF-B pools in hepatocytes. TNF␣ induces a coordinated proteolysis and resynthesis of IB isoforms to produce dynamic changes in NF-B nuclear abundance.Multicellular organisms have evolved mechanisms for the coordinate expression of inducible genes through ligand-dependent receptors. Ligand binding to high affinity receptors located on the plasma membrane generate second messenger signals that can influence the activity or abundance of transcription factors through post-translational modifications including signal-induced phosphorylation and/or proteolysis. Hormone-activated gene transcription plays an important role in many homeostatic processes, including the cytokine cascade for lymphocyte expansion (1), and the change in expression of liver genes in response to systemic inflammation known as the hepatic acute-phase response (APR 1 ; reviewed in Refs. 2-4).The APR is the consequence of inducible transcriptional activation of hepatic genes required for blood pressure regulation, such as angiotensinogen (2, 5), and those involved in macrophage opsonization and wound repair (6) through the effects of macrophage-derived interleukins-1, interleukin-6, and tumor necrosis factor ␣ (TNF␣) (6). Hepatocyte-specific transactivators modified during the APR include AP-1 (7), signal transducers and activators (8), nuclear factor-interleukin 6 (9), and nuclear-factor-B (NF-B) (10). The angiotensinogen gene is transcriptionally activated during the APR by the effect of a single regulatory element, the acute-phase response element (APRE) (11, 12). The APRE is a target for intracellular signaling initiated by the liganded TNF␣ type I receptor that activates latent DNA binding activity of the potent NF-B transcription factor fa...