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(Hypertension. 1996;27:1009-1017.)
© 1996 American Heart Association, Inc.

Articles

Tumor Necrosis Factor Activates Angiotensinogen Gene Expression by the Rel A Transactivator

Presented in part at the 67th Scientific Sessions of the American Heart Association, Dallas, Tex, November 14-17, 1994.

Allan R. Brasier; Junyi Li; Kenneth A. Wimbish

From the Departments of Medicine (A.R.B., J.L., K.A.W.) and Human Biological Chemistry and Genetics and the Sealy Center for Molecular Science (A.R.B.), University of Texas Medical Branch, Galveston.

Correspondence to Allan R. Brasier, Division of Endocrinology, MRB 3.142, University of Texas Medical Branch, 301 University Blvd, Galveston, TX 77555-1060.

	Abstract

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Abstract Angiotensinogen encodes the only known precursor of angiotensin II, a critical regulator of the cardiovascular system. Transcriptional control of angiotensinogen in hepatocytes is an important regulator of circulating angiotensinogen concentrations. Angiotensinogen transcription is increased by the inflammatory cytokine tumor necrosis factor (TNF)- by a nuclear factor–B–like protein binding to an inducible enhancer called the acute-phase response element. By gel mobility shift assays, we observe two specific acute-phase response element–binding complexes, C1 and C2. The abundance of C2 is not changed by TNF treatment. In contrast, C1 is faintly detected in untreated cells, and its abundance increases by fivefold after stimulation. We identify the nuclear factor–B subunits in these complexes using subunit-specific antibodies in the gel mobility "supershift" assay. The transcriptionally inert nuclear factor–B DNA-binding subunit NF-B1 is present in both control and stimulated hepatocyte nuclei. Its abundance changes weakly upon TNF stimulation. In contrast, the potent transactivating protein Rel A is not found in unstimulated hepatocyte nuclei and is recruited by TNF- into the C1 DNA-binding complex. Overexpression of Rel A results in acute-phase response element transcription. Cotransfection of a chimeric GAL4–Rel A protein with GAL4 DNA-binding sites is a strategy that allows for selective study of Rel A. The GAL4:Rel A chimera is a TNF-–inducible transactivator. Deletion of the amino-terminal 254 amino acids of Rel A produces a constitutive activator (that is no longer TNF- inducible). The cytokine induction of Rel A, then, is mediated through its amino-terminal 254 amino acids. We conclude that Rel A:NF-B1 is a crucial cytokine-inducible transcription factor complex regulating angiotensinogen gene synthesis in hepatocytes and may be involved in controlling the activity of the renin-angiotensin system.

Key Words: nuclear factor kappa B • renin-angiotensin system • angiotensinogen • acute-phase reaction

	Introduction

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The AGT gene encodes the only known glycoprotein precursor of the potent vasopressor Ang II and is transcriptionally activated in hepatocytes during the acute-phase response.^{1 2} Although AGT is expressed in a variety of tissues in which local AGT formation probably plays an important role in organ function,^{3 4} hepatic expression of AGT controls to a large extent the circulating levels of AGT within plasma. Because the hepatocyte lacks the ability to store presynthesized proteins, changes in AGT gene expression are directly coupled to changes in AGT protein secretion.^{5 6} These observations, in conjunction with numerous physiological and genetic studies indicating AGT concentrations to be rate-limiting for Ang II formation in the intravascular space,^{7 8 9 10 11 12} argue that transcriptional induction of hepatic AGT expression is an important control point for the activity of the intravascular RAS.

AGT gene expression in liver is tightly controlled through the influence of multiple hormonal stimuli, including (1) the steroid hormones: glucocorticoids,^{6 13 14 15 16} estrogens,^{3 17} and triiodothyronine^{18 19} ; (2) the peptide product of AGT processing, Ang II^{20 21 22 23} ; (3) the state of cellular differentiation^{24 25 26 27} ; and (4) cytokine hormones elaborated as a consequence of systemic inflammation (the APR^{1 2 28 29} ). These hormones alter AGT expression under physiological conditions by affecting the abundance of transcription factors that bind to the AGT promoter in hepatic nuclei.

One well-characterized physiological activator of AGT expression is the hepatic APR, a stereotypical response of the mammalian liver to the initiation of inflammation. In the APR, local injury or inflammation results in cytokine elaboration (IL-1 and TNF-); these hormones induce a switch in hepatic gene synthesis to producing proteins involved in macrophage opsonization and wound repair. The APR, initiated experimentally by intraperitoneal lipopolysaccharide and effected by the production of TNF-, is a potent inducer of hepatic AGT expression. Study of AGT regulation during the APR has revealed insights into transcriptional control elements and DNA-binding proteins that control expression of the rat AGT gene (reviewed in Reference 30). One crucial DNA control element, located between -531 and -557 in the rat gene 5' to the transcription start site, contains the sequence 5'-AGTTGGGATTTCCCAACC-3' that we have called the APRE. The APRE is a TNF-–inducible enhancer that confers TNF- induction onto an inert minimal promoter.¹

The APRE functions because it is a binding site for the potent transcription factor complex NF-B. NF-B is a multiprotein complex encoded by different genes but sharing a homologous 250–amino acid N-terminal DNA-binding domain. These members include Rel A (p65), Rel B, NF-B1 (NF-B p50), and NF-B2 (NF-B p49) (reviewed in Reference 31). Rel A is a powerful transactivating NF-B family member that binds to DNA sequences either as homodimers or as a heterodimer with one of the strong DNA-binding subunits, NF-B1 or NF-B2. In resting cells, NF-B is sequestered in the cytoplasm through reversible interactions with a family of inhibitory proteins called IB.^{32 33 34} Cytokines such as TNF- activate NF-B by disrupting the IB complex through a coupled phosphorylation/degradation step, allowing the NF-B complex to enter the nucleus, bind to inducible promoters, and stimulate their transcription.

Mutations of the APRE that disrupt NF-B binding also prevent cytokine induction of the AGT promoter.¹ That NF-B binds to the AGT APRE has been argued circumstantially on the basis of similar methylation-interference patterns, the existence of latent cytoplasmic APRE binding activity, and cross-competition experiments in in vitro DNA-binding assays.^{2 14} Characterization of the NF-B subunit composition that binds the AGT APRE is important because the heteromeric complexes Rel A:Rel A,³⁵ Rel A:Rel B,³⁶ Rel B:NF-B1,³⁷ and Rel A:NF-B1³⁸ have all been observed to bind to similar DNA sequences with distinct transactivational activities and modes of regulation. What are the relevant NF-B subunits controlling hepatic AGT expression?

In this article, we characterize the subunit composition of the NF-B members that bind the AGT APRE in control and TNF-–stimulated human hepatoblastoma (HepG2) nuclei. TNF- induces APRE transcriptional activity in a dose-dependent fashion. In parallel, TNF increases the DNA binding of an NF-B–specific complex of unique mobility. Using NF-B subunit–specific antibodies in the gel mobility "supershift" assay, we identify the NF-B subunits NF-B1 and Rel A as the relevant NF-B members controlling AGT expression.

	Methods

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Transfection and Cell Culture
The human hepatoblastoma cell line HepG2 was obtained from the American Type Collection Catalog and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 µg/mL penicillin, 100 µg/mL streptomycin, 0.025 µg/mL fungizone, 10 mmol/L nonessential amino acids, and 1 mmol/L sodium pyruvate in an atmosphere of 5% CO₂. Transient transfections were performed with the calcium phosphate technique^{14 39} in triplicate 60-mm plates using 10 µg luciferase reporter, 4 µg CMV–ß-Gal (internal control), the indicated CMV–Rel A expression plasmid, and carrier pGEM7Z plasmid to constitute a total of 20 µg for each set of triplicate plates. CMV–ß-Gal was chosen as an internal control because the CMV promoter is a high-level–expression promoter in HepG2 cells whose activity is not influenced by TNF- treatment. The 1.25 µg concentration is selected to produce galactosidase expression that accurately reflects changes in transfection efficiency. In the experiments in which transient overexpression is used, the expression plasmid is included in place of carrier pGEM DNA to maintain the same DNA concentration per plate. Twenty-four hours after transfection, cells were washed, fresh medium was added, and cells were cultured for an additional 20 hours. Four hours before harvest, recombinant human TNF- was added at the indicated concentrations.

Reporter Assays
Transfected cells were harvested by washing two times in PBS and lysed on the plate by the addition of 0.3 mL of lysis buffer (25 mmol/L Tris-phosphate, pH 7.8, 2 mmol/L DTT, 2 mmol/L 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid, 10% glycerol, 1% Triton X-100). After cells were incubated in lysis buffer for 15 minutes at room temperature, plates were scraped, and lysates were transferred to 1.5-mL Eppendorf centrifuge tubes and spun in a microcentrifuge at 10 000 rpm for 5 minutes. Cytoplasmic lysate (100 µL) was assayed for luciferase reporter activity by injection of 100 µL of luciferase assay reagent (20 mmol/L Tricine, 1.07 mmol/L [MgCO₃]Mg[OH]₂ · 5H₂O, 2.67 mmol/L MgSO₄, 0.1 mmol/L EDTA, 33.3 mmol/L DTT, 270 µmol/L coenzyme A, 470 µmol/L luciferin, 530 µmol/L ATP). Light output was measured over 16 seconds in a refrigerated Berthold 953 luminometer. ß-Gal activity was measured separately by 100 µL to the synthetic substrate ONPG, and quantification of the product was determined spectrophotometrically. Luciferase activity was determined by subtracting machine blank and normalizing each plate to ß-Gal activity. The mean and SD were then calculated for each independently transfected set of triplicate plates. Activity (in multiples) was calculated by dividing the normalized luciferase activity by that determined for the same reporter in the absence of TNF- stimulation.

Gel Mobility Shift Assays of Nuclear Extracts
To extract nuclear protein from treated cells, the cytoplasmic lysate was aspirated from the nuclear pellet, 50 µL of nuclear extract buffer (in mmol/L: HEPES 10, pH 7.8, KCl 400, EGTA 0.1, EDTA 0.1, PMSF 1, and DTT 1) was added, and nuclear protein was extracted by gentle agitation at 4°C to allow lysis of the nuclei and extraction of the DNA-binding proteins. The insoluble chromatin and membranes were removed by centrifugation for 5 minutes in a microcentrifuge at maximal speed (10 000 rpm), and the supernatant was saved for analysis of DNA-binding activity. Typically, we obtain nuclear protein at 2 to 5 µg/mL (representing 100 to 250 µg/60 min plates).

Gel mobility shift assays were performed as previously described,^{1 2 14 39} using the indicated amounts of nuclear extract incubated with 2x10⁴ cpm of duplex oligonucleotide (labeled by Klenow "fill-in" using [-³²P]dATP). After binding for 15 minutes at 22°C, samples were electrophoresed on 6% to 7% nondenaturing polyacrylamide gel in 1x TBE (25 mmol/L Tris, 25 mmol/L boric acid, and 0.5 mmol/L EDTA) at 180 V (constant voltage) for 2 hours. Competition was performed by the addition of excess nonradioactive double-stranded oligonu-cleotide competitor at the time of addition of radioactive probe. The sequences of the APRE are as follows: APRE WT GATCCACCACAGTTGGGATTTCCCAACCTGACCA

GAGGTGTCAACCCTAAAGGGTTGGACTGGTCTAG

*** APRE M6 GATCCACCACAGTTGTGATTTCACAACCTGACCA

GTGGTGTCAACACTAAAGTGTTGGACTGGTCTAG APRE M2 GATCCACCACATGTTGGATTTCCGATACTGACCA

GTGGTGTACAACCTAAAGGCTATGACTGGTCTAG (Asterisks indicate points of NF-B contact using the methylation interference assay.¹ )

After electrophoresis, gels were dried and exposed overnight at -70°C to Kodak X-AR film or for quantification to a PhosphorImager cassette and analyzed by ImageQuant software with a Molecular Dynamics 425E PhosphorImager.

Antibody supershift assays were performed by addition to the binding reaction of 1 µL of unfractionated antibody in serum and incubation for 20 minutes at 22°C. The antibody:NF-B:DNA complexes were electrophoresed on a softer polyacrylamide gel (5%); the free probe and bromphenol blue tracking dyes were electrophoresed off the gel to allow visualization of the antibody:NF-B:DNA complexes. Anti–NF-B1 antibody was produced by immunizing rabbits with a bacterially expressed polyhistidine-tagged NF-B1 protein. This NF-B1 protein was produced by use of the T7 promoter/polymerase in Escherichia coli and purified to homogeneity by nickel-agarose (Ni-NTA, Qiagen) affinity chromatography. The NF-B1 domain is a unique region in the C-terminus not conserved with other NF-B family members (amino acids 410-480). This antibody binds and supershifts recombinant full-length NF-B1 protein. Anti–Rel A, anti–c-Rel, and anti–NF-B2 antibodies were obtained commercially (Santa Cruz Biotech). Anti–Rel A does not cross-react with recombinant NF-B1 and recognizes a 65-kD protein in HepG2 nuclear extracts, and thus is subunit specific.

Plasmid Construction
APRE-LUC consists of the trimerized rat AGT APRE WT sequences ligated through BamHI/Bgl II ends into the p59 rat AGT minimal promoter driving the expression of the firefly luciferase reporter gene.^{1 2 14 26 40} Site mutations of the rat AGT APRE were constructed by the same strategy.

The eukaryotic expression vector encoding full-length human Rel A was produced by ligating the 2.2-kb coding sequences (1-551) of Rel A into the BamHI site of the eukaryotic expression vector pcDNAI (InVitrogen Inc). This expression vector produces Rel A mRNA under the direction of the powerful eukaryotic CMV long-terminal repeat. Dideoxynucleotide sequencing using the SP6 primer site was used to confirm its authenticity. The GAL4–Rel A expression vector was constructed by use of the eukaryotic expression vector pSG424⁴¹ that produces GAL4 (1-147) under control of the SV40 early region promoter/enhancer. The multiple cloning site was altered to place a BamHI restriction site in frame with the GAL4 coding sequences (pGAL4 Ad). Rel A (1-551) was then cloned as a BamHI fragment into the pGAL4 Ad plasmid. Rel A (255-551) was produced by amplifying in the polymerase chain reaction the Rel A (255-551) coding sequences, restricting the PCR fragment with BamHI and Xba I, and ligating into pGAL4 Ad restricted with BamHI and Xba I. The upstream oligonucleotide used for the Rel A amplification was 5'-GCC ATT GAA TTC T CTA GATTAG GAG CTG ATC TGA CTC AGC AGG-3' (the Xba I site is underlined and stop codon indicated in bold). Sequencing of the GAL4–Rel A coding sequences was performed to ensure authenticity of the coding sequences. The UAS-LUC reporter plasmid was constructed by use of the tandem GAL4 binding sites ligated upstream from the -59 rat AGT minimal promoter. The UAS sequences are GATCCCGGAGGACTGTCCTCCGCGGAGGACTGTCCTCCGA and GGCCTCCTGACAGGAGGCGCCTCCTGACAGGAGGCTCTAG. All plasmids were prepared on cesium chloride gradients before transfection, and their concentrations were determined spectrophotometrically.

	Results

Top Abstract Introduction Methods Results Discussion References

 
To study induction of the NF-B proteins, we constructed a sensitive reporter plasmid for measuring the transcriptional activation of the APRE. The plasmid consists of three copies of the rat AGT APRE ligated upstream from the -59 rat AGT promoter in the sensitive luciferase reporter gene (APRE-LUC). APRE-LUC has previously been demonstrated to initiate transcription at the nucleotide (nt) +1 cap site in exon 1 of the rat AGT gene.² APRE-LUC was transfected into HepG2 cells along with internal control plasmid CMV–ß-Gal, an activity that is independently measured to normalize for plate-to-plate changes in luciferase reporter activity; transfectants were stimulated for 4 hours (44 hours after transfection) with the indicated increasing concentrations of recombinant human TNF-, and reporter activity was measured. Fig 1 summarizes the mean±SD of three independent transfections. Relative to unstimulated control, TNF- increased APRE transcriptional activity 7.5-fold at 0.3 ng/mL to 27-fold at 30 ng/mL. Mediation of the TNF- induction by specific DNA sequences on the APRE is indicated by measurement of the TNF- induction of APRE site mutations (APRE M6 and APRE M2) ligated into the same promoter. As shown in Fig 1, reporter gene activity produced by either APRE M6-LUC or APRE M2-LUC is inert to TNF- stimulation.

View larger version (19K): [in this window] [in a new window]   Figure 1. Bar graph showing that TNF- induces AGT APRE transcriptional activity in transient transfections of HepG2 cells. Human HepG2 cells were transiently transfected with reporter genes driven by APRE DNA binding sites and site mutations ligated upstream of the rat AGT gene promoter. Twenty-four hours after transfection the medium was changed, and cells were cultured for an additional 20 hours. Four hours before harvest, triplicate plates of cells were stimulated with recombinant human TNF- at the indicated concentrations. Data represent the mean±SD of three independent transfections of independently assayed luciferase and internal control ß-Gal reporter activities. At 30 ng/mL, TNF- activates APRE WT (open bars) reporter activity 25-fold, whereas the site mutations of the APRE, APRE M6 (solid bars), and APRE M2 (hatched bars) are not reproducibly TNF- inducible.

To identify the TNF-–inducible APRE binding proteins, nuclear extracts from control and TNF-–stimulated HepG2 cells were prepared and analyzed by EMSA. In the absence of TNF-, a strong DNA-binding complex (C2) is detectable, and a slower mobility complex (C1) is faintly detectable (Fig 2). Upon TNF- treatment, the slower-mobility C1 complex increases its binding to the APRE in a dose-dependent fashion (see also Fig 3). Binding specificity of the TNF-–inducible C1 complex is shown in Fig 3, in which unlabeled oligonucleotides containing site mutations with the APRE are included in the binding assay as competitors. Inclusion of unlabeled APRE WT DNA as a competitor but not the site mutations APRE M6 or APRE M2 DNA inhibits both the constitutive C2 complex and the TNF-–inducible C1 complex, indicating that both C1 and C2 complexes have "NF-B–like" DNA-binding specificity.

View larger version (59K): [in this window] [in a new window]   Figure 2. TNF- induces AGT APRE DNA-binding activity in a dose-dependent fashion. Human HepG2 cells were stimulated with TNF- at the indicated concentrations for 4 hours before the harvest and extraction of nuclear protein according to our previously published techniques.39 Shown is an autoradiogram of a 7% EMSA using radiolabeled APRE in the presence of 2 µg poly dI-dC as a nonspecific competitor and 15 µg nuclear protein. Two nucleoprotein complexes are seen, labeled complex 1 (C1) and C2. C2 is the predominant complex in untreated control cells (C1 is only faintly detectable). TNF- induces the abundance of C1 in a dose-dependent manner. The abundance of C2 does not change with TNF- treatment (see also Fig 3).

View larger version (88K): [in this window] [in a new window]   Figure 3. The TNF-–inducible DNA-binding complex binds the APRE in a sequence-specific fashion. Forty micrograms of nuclear extract from unstimulated and TNF-–stimulated HepG2 cells was used in the EMSA in the absence or presence of unlabeled APRE DNA binding competitor DNA. Shown is the autoradiogram with radiolabeled APRE WT DNA. Here and in Fig 4, poly dA-dT was used as a nonspecific competitor to abolish the binding of a nonspecific complex that closely comigrates with C2. Both C1 and C2 are completely competed with a 100-fold excess of unlabeled APRE WT DNA but not APRE M6 or APRE M2 DNA-binding sites. These observations indicate that the constitutive C2 complex and the TNF-–inducible C1 complex both bind DNA in a sequence-specific manner.

Gel mobility "supershift" assays were used to identify which NF-B subunits are contained within the C1 and C2 nucleoprotein complexes. This assay relies on NF-B subunit–specific antibodies to produce a more slowly migrating supershift antibody/NF-B/DNA complex that allows for the unambiguous identification of that protein in the DNA complex. In Fig 4, the antibody supershift assay was used on control (unstimulated) or TNF-–stimulated HepG2 cell nuclear extracts. In control nuclear extracts, the APRE binding complexes were not affected by the addition of preimmune antibodies or antibodies directed against NF-B2 (p49), c-Rel (Rel B), or Rel A (p65) subunits. However, addition of anti–NF-B1 antibodies produced an additional supershifted complex, indicating that NF-B1 protein is the predominant NF-B protein contained within the C2 complex in unstimulated HepG2 cells. The intensity of supershifted NF-B1 increases with TNF- treatment. In control nuclear extracts, addition of anti–Rel A antibodies does not produce a detectable supershifted complex, indicating that the nuclear DNA binding activity of Rel A is low. In contrast, anti–Rel A antibodies produce a strong supershift in the TNF-–stimulated nuclear extracts containing the C1 complex, indicating that the C1 nucleoprotein complex contains Rel A. We interpret these studies as indicating that NF-B1 is the predominant NF-B subunit binding the APRE (C2 complex) in unstimulated HepG2 nuclei and that DNA-binding activity of Rel A, and to a lesser extent of NF-B1, is induced in HepG2 nuclei in response to TNF- treatment.

View larger version (70K): [in this window] [in a new window]   Figure 4. Gel mobility "supershift" assay demonstrates NF-B subunits within the APRE nucleoprotein complex. Nuclear extracts from unstimulated and TNF-–stimulated HepG2 cells were used in the gel mobility supershift assay with radiolabeled APRE DNA in the presence of subunit-specific NF-B antibodies against NF-B1 (p50), NF-B2 (p49), c-Rel (Rel B), and Rel A (p65). This assay will produce an additional slower-mobility supershift band composed of antibody:NF-B subunit:DNA complex. In this assay, a 5% EMSA was used to visualize the supershift bands, and the free probe was electrophoresed off the gel, resulting in a smeared DNA complex. As with preimmune serum (PI), the addition of anti–NF-B2 and c-Rel antibodies do not perturb the nucleoprotein complex. The addition of anti–NF-B1 antibodies produces an additional complex both in the control and TNF-–stimulated extracts. The intensity of the supershifted NF-B1 increases slightly with TNF- treatment. Addition of anti–Rel A antibodies produces a supershift complex only in the TNF-–stimulated extracts (containing C1). No Rel A supershifting is seen in untreated nuclear extracts. These observations indicate that the major effect of TNF- is to induce the abundance of Rel A binding activity in HepG2 nuclei.

Previous studies indicate that NF-B1 and Rel A have distinct transactivation potentials,^{22 42 43 44} with NF-B1 being a transcriptionally inert DNA-binding protein and Rel A being a potent transcriptional activator. To demonstrate that the activator Rel A is involved in the stimulation of the AGT APRE, we transiently overexpressed Rel A in the HepG2 transfection assay using APRE-LUC as a reporter gene. Fig 5 demonstrates the mean±SD (of three independent experiments) of transfections using increasing amounts of a eukaryotic expression vector producing Rel A under the direction of the potent CMV long-terminal repeat (pcDNA–Rel A). Using this potent expression vector, we can achieve levels of Rel A high enough to saturate available IB, allowing Rel A to function as a constitutive activator. Overexpression of pcDNA–Rel A results in a dose-dependent increase in luciferase reporter activity. Consistent with earlier reports, in separate experiments we have tested the ability of an NF-B1 expression vector to activate the APRE-LUC reporter and were unable to stimulate its activity (not shown).

View larger version (21K): [in this window] [in a new window]   Figure 5. Bar graph showing that transient overexpression of Rel A activates AGT APRE in HepG2 cells. The NF-B subunit Rel A was transiently overexpressed in the presence of the APRE/p59ratLuc reporter plasmid. Forty-eight hours after transfection, cells were harvested for independent assay of luciferase and internal control ß-Gal activity (data represent the mean±SD of three independent experiments). The pcDNA–Rel A expression vector activated APRE/p59ratLuc reporter activity in a dose-dependent fashion sevenfold. In this experimental paradigm, Rel A constitutively transactivates the APRE because its expression saturates the IB inhibitor. These data indicate that Rel A is a potent activator of APRE transcriptional activity.

Transient overexpression of Rel A using APRE-linked reporter genes cannot distinguish the individual contributions of the various NF-B family members because both NF-B1 and Rel A bind to the APRE (Figs 2 through 4). To isolate the study of Rel A, we constructed a eukaryotic expression vector that encodes Rel A protein fused in frame to a DNA-binding domain of a yeast transcription factor (GAL4) that is not expressed in mammalian cells. This chimeric protein, GAL4-Rel A, will uniquely bind to GAL4 binding sites (called UAS) and allows for the isolated study of the transcriptional properties of Rel A in response to TNF-. The strategy of fusing GAL4 DNA-binding domains to other transactivators is a standard technique that has been applied to study members of other transcription factors individually.^{41 45 46} The concept is schematically diagrammed in Fig 6A. Fig 6B demonstrates the results of a transient transfection assay in which the GAL4–Rel A expression vector is cotransfected with UAS-LUC reporter genes into HepG2 cells. In the presence of the full-length Rel A protein (1-551), a dose-dependent increase in UAS-LUC reporter activity is measured. To identify the domain of Rel A that is responsive to TNF-, we deleted the sequences coding the N-terminal 255 amino acids of the Rel homology domain and fused this coding sequence to the GAL-4 DNA binding domain GAL4-Rel A (255-551). This protein contains the activation surfaces of the Rel A transcription factor but lacks the DNA-binding and IB-interactive domains. In parallel transient transfection experiments, the GAL4–Rel A (255-551) was a potent activator of UAS-LUC reporter activity but was no longer TNF-–inducible. Taken together, these data demonstrate not only that Rel A (1-551) is a TNF-–inducible activator but also that the N-terminal DNA-binding and IB interaction domain is absolutely required for the effect.

View larger version (33K): [in this window] [in a new window]   Figure 6. A chimeric GAL4:Rel A protein is a TNF-–inducible transactivator. A, Schematic view of the GAL4:Rel A protein. The similar DNA-binding characteristics of the NF-B proteins preclude their isolated study. To circumvent this problem, a chimeric protein of Rel A fused to the 147–amino acid yeast DNA-binding domain of the GAL4 transcription factor (not expressed in mammalian cells) is constitutively expressed in transiently transfected hepatocytes. Transcriptional activity of the GAL4:Rel A protein can then be selectively measured by basal and TNF-–stimulated activity of the GAL4 DNA-binding sites (UAS-LUC). Top, Schematic view of the GAL4:Rel A (1-551) chimera in unstimulated hepatocytes. The IB interactive domain of the Rel A molecule inactivates the protein in the absence of hormonal stimulation. Bottom, GAL4:Rel A chimera after TNF- stimulation. TNF-, upon interacting with the 55-kD TNF receptor (TNFR), results in disruption of the Rel A:IB complex. The GAL4:Rel A protein then enters the nucleus, where the GAL4 DNA-binding domain recognizes the UAS-linked luciferase reporter and stimulates its transcription. B, Bar graph showing that GAL4:Rel A transcriptional activity is activated by TNF-. HepG2 cells were transiently transfected with GAL4:Rel A expression vectors and the UAS-LUC reporter. Forty-four hours after transfection, cells were stimulated for 4 hours in the absence or presence of increasing concentrations of recombinant human TNF- (at the indicated doses). UAS luciferase reporter activity was increased 4.5-fold in a dose-dependent fashion by TNF- relative to untreated controls in response to the full-length Rel A (1-551) fused to GAL 4. Deletion of the 254 N-terminal amino acids of Rel A [GAL 4–Rel A (255-551)] resulted in a constitutive activator of GAL4 binding sites not further inducible by TNF- (data represent the mean±SD of three independent experiments).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Transcriptional regulation of the AGT gene in hepatocytes influences the activity of the intravascular RAS. In earlier studies, we identified a multihormone-responsive enhancer in the 5' flanking region of the rat AGT gene between -615 and -470 that integrates hormonal signals from both glucocorticoids and cytokines. Within this enhancer, one element is a bona fide cytokine-inducible enhancer (the APRE) that is absolutely required for TNF- or IL-1 induction of AGT gene expression. Moreover, this enhancer is a high-affinity DNA-binding site for BPi. In previous studies, this association was argued on the basis of multiple lines of evidence: (1) The BPi produced a methylation interference pattern on the APRE identical to that produced by purified NF-B from bovine spleen; (2) both BPi and NF-B recognized canonical NF-B binding sites; (3) BPi and NF-B were both activated in hepatocytes by phorbol esters in the absence of new protein synthesis; and (4) BPi and NF-B could be identified in a sequestered form in hepatocyte cytoplasmic extracts.

The demonstration that the multiprotein NF-B complex has distinct transcription properties based on its subunit association^{35 36 37 38} demands that these subunits be identified for a complete understanding of AGT gene expression in hepatocytes.

In this article, we report the identification of the constitutive and TNF-–inducible NF-B subunits that bind to the APRE. NF-B subunit–specific antibodies used in the supershift assay demonstrate that NF-B1 binds the APRE in both resting and TNF-–stimulated nuclei. Its abundance is only slightly changed by TNF- treatment. In contrast, Rel A binding activity is not detectable in unstimulated nuclear extracts and is strongly induced by TNF-. Rel A is a potent activator of APRE transcription, as shown by transient overexpression assays using the APRE-linked luciferase reporter gene. That Rel A is TNF-–inducible is argued not only by the gel mobility supershift assays but also by transcriptional analysis of GAL4:Rel A chimeras in response to TNF-. Moreover, we demonstrate that the N-terminus of Rel A (amino acids 1-254) is absolutely required for TNF- induction, because its deletion abolishes TNF- inducibility and results in a potent constitutive activator of GAL4 binding sites.

The NF-B family is regulated through multiple protein interactions that modify their transcriptional and DNA-binding activities. NF-B1 is an inert DNA-binding protein representing an N-terminal proteolytic product of a 105-kD precursor as a consequence of processing from the ubiquitin-proteasome pathway.^{47 48} NF-B1 can exist in a cytoplasmic location in certain cell types, apparently depending on the relative expression of cytoplasmic Rel A, with which NF-B1 dimerizes efficiently, and the oncoprotein BCl-3, a nuclear protein that alters the activity and subnuclear distribution of NF-B1.^{49 50} In HepG2 cells, our supershift assays clearly demonstrate that NF-B1 DNA binding is present in the nuclei of unstimulated cells. On the basis of comparison of the APRE sequence with optimally defined NF-B binding sites produced by PCR-based binding site selection strategy of random oligonucleotides, the APRE represents a predicted high-affinity NF-B1 binding site. Consistent with earlier reports of NF-B1 as a transcriptionally silent DNA-binding protein,^{31 42 47} we are unable to transactivate the APRE by overexpressing NF-B1 alone. Thus, NF-B1 binding is transcriptionally silent on the AGT promoter.

Our data indicate that the C2 complex is probably composed of homodimeric NF-B1, whereas the C1 complex is probably composed of NF-B1:Rel A heterodimers. This statement is based on the following points. (1) Both C1 and C2 bind DNA with NF-B specificity. (2) NF-B1 is the only supershifted NF-B subunit in unstimulated nuclei in which the C2 complex strongly binds the APRE. (3) The abundance of supershifted NF-B1 increases after TNF- treatment, whereas the amount of complex C2 does not change (Fig 3), indicating that NF-B1 is probably contained within the C1 complex. (4) Recombinant Rel A (as a homodimer) does not bind with high affinity to the APRE (data not shown) and probably requires NF-B1 to target it to the APRE. We note that APRE transcription occurs parallel with the appearance of the C1 nucleoprotein complex. Taken together, the weight of the evidence argues that Rel A is the relevant APRE transactivator in response to TNF- treatment.

The potent transcriptional activator Rel A is encoded by a separate gene but is related to NF-B1 by amino acid homology in the first 250 amino acids (called the Rel homology domain), a region first described in the C-Rel proto-oncogene and the drosophila morphogen dorsal.^{31 47} In resting cells, Rel A is sequestered in the cytoplasm through reversible interactions with IB proteins³² ; one IB protein identified in rat liver is the homologue of human MAD3 (IB-).⁵¹ IB contacts dimeric Rel A through a conserved domain with erythrocyte ankyrin, inactivates its DNA-binding activity, and masks its nuclear localization signal.⁵² More detailed study will be required to identify how the N-terminal Rel homology domain confers TNF- induction. This domain is involved in DNA binding, interaction with IB, nuclear localization, and dimerization and also contains protein kinase A and protein kinase C phosphoacceptor sites.^{53 54 55} In the GAL4:Rel A chimera, the GAL4 DNA-binding domain supplies DNA binding, nuclear localization, and dimerization activity for the fusion protein. Thus, we believe that the most likely mechanism for loss of TNF- induction is that amino acids 1-254 are necessary for IB interaction. The deletion of the Rel homology domain prevents sequestration of Rel A in the cytoplasm, allowing Rel A to enter the nucleus independent of the TNF- hormone and stimulate reporter gene activity. Consistent with this view, we note that the activity of GAL4:Rel A (1-551) is less than GAL4:RelA (255-551) in control (non–TNF-–stimulated) HepG2 hepatocytes. Immunofluorescence assays of transfected HepG2 cells using an antibody specific for the chimeric protein will be important to demonstrate this mechanism.

Recruitment of Rel A, the NF-B subunit with potent transactivation properties, into the APRE:NF-B1 complex is vital to the process of transactivation. In COS cells and T lymphocytes, Rel A contains a potent C-terminal transactivating region necessary for promoter activation.^{44 54} The observation that GAL4:Rel A (255-551) is a constitutive activator of GAL4 binding sites indicates that the location of the Rel A transactivating domain is also within the C-terminus in HepG2 cells. The C-terminal transactivating region of Rel A is composed of an acidic amino acid residue–rich region contained in a putative amphipathic -helix.⁴⁴ This Rel A acidic activator domain may be necessary to recruit coactivator binding onto the AGT promoter. We also note that Rel A:NF-B1 bends DNA upon binding,⁵⁶ which perhaps could result in either promoter looping or alterations in AGT chromatin structure. The distinction among these various mechanisms will require additional study.

How is Rel A regulated by TNF-? Although the mechanism will need to be validated for hepatocytes, we believe that our data are consistent with the general "working model" of TNF- action based on studies in T lymphocytes and other cell lines. These studies have shown that TNF- activates Rel A by disrupting the Rel A:IB complex; this allows the potent Rel A transactivator to enter the nucleus and stimulate transcription. Activation of the TNF receptor results in activation of phosphatidyl-choline–specific phospholipase C and production of 1,2-diacylglycerol. 1,2-Diacylglycerol in turn activates an acidic sphingomyelinase that is thought to be the primary regulator of the Rel A:IB complex through a ceramide-activated kinase or phosphatase activity.^{57 58 59} The IB molecule is then directly phosphorylated and proteolyzed.^{60 61 62} Thus, TNF- activates Rel A through a posttranslational modification of its inhibitory subunit, resulting in the nuclear appearance of Rel A (schematically diagrammed in Fig 7). Indeed, we have previously shown that induction of NF-B DNA-binding activity is independent of new protein synthesis.^{1 40}

View larger version (16K): [in this window] [in a new window]   Figure 7. Schematic model of TNF- activation of AGT APRE transcriptional activity through the Rel A:NF-B1 heterodimer. NF-B1 homodimers are the predominant APRE binding proteins in unstimulated HepG2 cells. Rel A:NF-B1 heterodimers are activated in the cytoplasm of HepG2 hepatoblastoma cells through interactions with the inhibitory (IB) proteins. TNF- activates nuclear translocation of Rel A:NF-B1 complexes by disruption of the NF-B:IB complex. The Rel A:NF-B1 heterodimers then associate with the AGT APRE and stimulate its transcriptional activity.

Our studies have focused on mechanisms of AGT gene activation by inflammation, particularly as a consequence of the hepatic acute-phase response. Why is AGT an acute-phase reactant? Overwhelming bacterial infection is a known hypotensive insult; for example, bacterial endotoxin (lipopolysaccharide) administration in humans produces circulatory collapse.⁶³ In this setting, AGT synthesis would be a homeostatic mechanism as a source for Ang II production in the setting of increased AGT metabolism. AGT may have functions other than serving as an intravascular reservoir for the Ang II peptides. We note that AGT is expressed at extremely high concentrations in fat depots^{3 64 65} and that TNF- is a potent regulator of fat metabolism.²⁵ In particular, local production of TNF- may have important roles in regulating lipolysis, insulin sensitivity, and AGT expression in the "local" RAS in fat depots.^{66 67}

Is TNF- activation of NF-B a relevant mechanism for other physiological mediators of AGT expression? One obvious example is the physiology of CHF. CHF is a condition in which hypoperfusion results in the pathophysiological activation of the RAS and the resultant peripheral vasoconstriction and diminished glomerular filtration rate by the vasoactive effects of Ang II. In several studies, circulating TNF- levels are increased in patients with CHF, and the magnitude of increase is predictive of short-term mortality.^{68 69} We speculate that in CHF, TNF- activation of AGT expression may be, in fact, sustaining Ang II production. Finally, NF-B (Rel A) is activated by multiple second messengers, including activators of protein kinase C, free radicals, IL-1, and UV light.⁴⁷ Our demonstration that Rel A is an AGT activator may implicate these other agents as modifiers of circulating or local RAS function. These latter questions are now directly approachable experimentally.

	Selected Abbreviations and Acronyms

 

ß-Gal = ß-galactosidase AGT = angiotensinogen Ang II = angiotensin II APR = acute-phase response APRE = acute-phase response element BPi = TNF-–inducible APRE DNA-binding protein complex of the NF-B class C1 = TNF-–inducible APRE DNA-binding complex CHF = congestive heart failure CMV = cytomegalovirus EMSA = electrophoretic gel mobility shift assay IL = interleukin NF-B = nuclear factor–B NF-B1 = p50 subunit of NF-B RAS = renin-angiotensin system Rel A = p65 subunit of NF-B TNF- = tumor necrosis factor- UAS = upstream activating sequences WT = wild-type

	Acknowledgments

 
This study was supported by National Institutes of Health grant 1R29 HL-45500 to Dr Brasier. Dr Brasier is an Established Investigator of the American Heart Association. We thank Rebecca Soliz for expert secretarial assistance.

Received May 16, 1995; first decision July 11, 1995; accepted August 14, 1995.

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