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(Hypertension. 1996;27:465-475.)
© 1996 American Heart Association, Inc.

Articles

Mechanisms for Inducible Control of Angiotensinogen Gene Transcription

Allan R. Brasier; Junyi Li

From the Department of Internal Medicine and Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston.

Correspondence to Allan R. Brasier, 3.142 Medical Research Building, 301 University Blvd, Galveston, TX 77555-1060. E-mail abrasier%intmeds1@mhost.utmb.edu.

	Abstract

Top Abstract Introduction Mechanisms of AGT Gene... Mechanisms of AGT Gene... Transcriptional Mechanism of the... Summary References

 
Abstract 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-. 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-B 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-B 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-B transcription factor provides a point for "cross talk" between angiotensin- and cytokine-activated second messenger pathways.

Key Words: nuclear factor-B • angiotensinogen • nuclear factor-IL6

	Introduction

Top Abstract Introduction Mechanisms of AGT Gene... Mechanisms of AGT Gene... Transcriptional Mechanism of the... Summary References

 
Inducible control of gene expression can be affected by changes in transcription rates, mRNA stability and processing, and in selected situations, translational initiation rates. Of these, changes in transcriptional initiation rates have been observed to be a common and important mechanism in controlling genes expressed by the mammalian hepatocyte. The liver, representing the major site for plasma protein biosynthesis and an organ that lacks the ability to store presynthesized proteins, primarily translates and secretes proteins through the constitutive pathway.¹ Therefore, hepatic stimuli that influence the expression of plasma protein genes also alter their secretory rates. Such coupling of gene expression with protein secretion has been documented for many plasma proteins, notably albumin and AGT.^{2 3}

Despite the close correlation between gene expression and protein secretion, the hepatocyte should not be viewed as a passive cell constitutively secreting proteins but rather as an exquisitely hormone-responsive target for the actions of circulating steroid and peptide hormones. In fact, under appropriate physiological stimuli, such as the hepatic APR, networks of inducible plasma protein genes can be activated, and different networks of genes can be silenced to respond to the anticipated physiological needs of the organism.⁴

Control of AGT biosynthesis is particularly relevant to the activity of the RAS. Generation of Ang I is the rate-limiting step in the formation of the potent pressor Ang II, the major effector peptide of the activated RAS. The only known glycoprotein precursor of Ang I, AGT, circulates at concentrations (0.6 µmol/L) that are rate limiting for its maximum velocity of formation within the intravascular space (the K_d of renin is 1 µmol/L).⁵ Conditions that alter circulating AGT concentrations therefore influence intravascular RAS activity.^{6 7} This idea is supported by many independent genetic and physiological studies, in which perturbations in AGT synthesis result in Ang II–dependent changes in blood pressure.^{6 7 8 9 10 11 12 13 14 15} Perhaps the most compelling evidence for the relationship of AGT gene expression and blood pressure is the effect of AGT gene dose on blood pressure in rodents. Gene targeting (homologous recombination) techniques to disrupt or duplicate AGT locus has allowed the generation of mice having various numbers of functional AGT alleles.¹⁰ In these mice, plasma AGT levels increase progressively in mice containing zero to four copies of the AGT locus, and concurrently a blood pressure increase of 8 mm Hg per gene copy is observed. These data provide a strong causal relationship between AGT genotypes and blood pressure.

Numerous physiological mediators have been described that control the expression of the AGT gene; from these observations, it becomes clear that AGT gene expression is under coordinate developmental, tissue-specific, and hormonal control. In rodents, where its expression has been most thoroughly investigated, AGT is expressed widely, for example, in the brain, heart, kidney, and adipose tissue, where its expression and processing may affect local organ function^{16 17 18} (reviewed in Reference 19). Within these tissues, differentiating events regulate AGT expression. This mechanism is exemplified by the robust postpartum rise in hepatic AGT synthesis²⁰ and by the in vitro differentiation of preadipocytes to mature adipocytes.^{21 22 23} Moreover, within organs normally expressing AGT, AGT transcription is responsive to diverse hormonal mediators.^{16 18 24 25} These agents include circulating steroid hormones (glucocorticoids,^{24 25 26} estrogens,^{27 28} and triiodothyronine²⁹ ); cytokine hormones, such as interleukin-1 or TNF-^{30 31 32 33 34 35} ; and acutely, Ang II.^{36 37 38 39 40} Intravascular Ang II production, generated as a consequence of RAS activation, regulates the synthesis and secretion of components necessary for the first rate-limiting step in Ang II production (Fig 1), upregulating AGT expression and downregulating renin secretion. Glucocorticoid excess, estrogen administration, and the malignant phase of essential hypertension are all associated with both elevated AGT levels and Ang II–dependent hypertension. Understanding how these factors control AGT synthesis is important to elucidating the pathophysiology of these hypertensive states. Ultimately, this knowledge may yield new therapeutic targets for blood pressure control.^{6 7}

View larger version (17K): [in this window] [in a new window]   Figure 1. Feedback regulatory mechanisms of the activated RAS. The liver represents the major source for circulating AGT in plasma. In the presence of renin, the first rate-limiting step in Ang II formation occurs with the N-terminal hydrolysis of the decapeptide Ang I from the AGT precursor. Apart from its direct vasoconstrictive and sodium-retentive effects, Ang II production regulates renin secretion negatively (the "short feedback loop")41 and stimulates hepatic AGT synthesis (the "positive feedback loop").37 38 39 ALDO indicates aldosterone.

We review the mechanisms for transcriptional activation of inducible AGT gene expression, focusing on the identification of relevant transcription factors involved. Our central concept is that inducible AGT gene expression is controlled by changes in the nuclear concentration of transcription factors. The induced DNA binding proteins recognize their cis-regulatory element through modular, highly conserved domains. Once bound to DNA control regions within selected promoters, inducible transcription factors regulate the rate of transcript formation by multiple mechanisms, including changing local chromatin structure and recruiting coactivator proteins that result in productive protein-protein interactions with the basal transcription machinery.^{42 43} We hypothesize that transcriptional control mechanisms of the AGT promoter in hepatocytes are important controllers of RAS activity.

	Mechanisms of AGT Gene Transcription by Glucocorticoids

Top Abstract Introduction Mechanisms of AGT Gene... Mechanisms of AGT Gene... Transcriptional Mechanism of the... Summary References

 
The most potent pharmacological activators of AGT gene expression are glucocorticoids. Administration of natural and potent synthetic glucocorticoids to humans increases circulating AGT⁷ ; in experimental animals, glucocorticoid administration induces accumulation of AGT mRNA transcripts in the liver.^{3 16 25 44} Conversely, adrenalectomized animals have suppressed levels of circulating AGT.⁴⁵ The single copy gene encoding AGT has been isolated from rodents and humans.^{46 47 48 49} On the basis of comparisons of the coding sequences and analysis of intron/exon structure, AGT was identified as a member of the serine protease inhibitor family, which includes ₁-antitrypsin.^{46 49 50} The first 700 base pairs of the rodent genes are sufficient for tissue-specific and hormonally regulated expression.^{26 32 44 51} Nuclear run-on assays and gene-transfer studies have shown that glucocorticoids exert their effects at the transcriptional level.^{24 26} Moreover, the fact that their induction of AGT gene expression is dose dependent, saturable, and antagonized by the antiglucocorticoid RU486 indicates that this transcriptional event is indeed mediated by the GR^{3 24 25 26 52}

Within the first 700 base pairs of the rodent AGT gene is a near-palindromic GRE containing the sequence 5'-AGAACATTTTGTTTC-3' (-584 to -570, Fig 2A). Transfection studies on reporter genes containing AGT promoters mutated at specific sequences indicated that GRE I is essential for glucocorticoid induction (Fig 2B). Site-directed mutation of GRE I into a sequence that did not bind recombinant GR abolished glucocorticoid induction of the reporter gene. GRE II, a hexameric "half-site" sequence located between -472 and -477, is insufficient for glucocorticoid induction in the absence of a functional GRE I. GRE II, however, is required for maximal glucocorticoid inducibility of the AGT promoter because its mutation attenuates but does not abolish glucocorticoid induction in the presence of wild-type GRE I. Both GRE I and GRE II bind to recombinant GR in in vitro DNA binding assays. Multimeric GRE I or GRE II confers glucocorticoid induction onto an inert reporter gene. On the basis of these data, we proposed a model in which GREs in the AGT promoter are hierarchical (GRE I is a dominant activity) and synergistic (both GREs stimulate greater than either GRE alone). This mechanism of regulation is shared with other liver genes, such as tyrosine aminotransferase, in which multiple GREs function to activate tyrosine aminotransferase transcription synergistically.⁵⁴

View larger version (25K): [in this window] [in a new window]   Figure 2. GREs in the multihormone-inducible enhancer are hierarchical and synergistic. A, Site-directed mutagenesis of the AGT promoter GREs. Rat AGT gene flanking sequences spanning nucleotides -615 to -470 are displayed; wild-type and mutant sequences were ligated upstream of the AGT minimal promoter in the luciferase reporter vector. GRE mutations (GRE Ms) were substitutions at GR contact points that block the ability of recombinant GR to bind. B, GRE I and GRE II mutations were tested in isolation and combination for glucocorticoid induction of luciferase reporter activity (normalized to cotransfected alkaline phosphatase reporter activity to control for plate-to-plate changes in transfection efficiency). Data are presented as normalized luciferase/alkaline phosphatase activity, and -fold activity is calculated by dividing normalized luciferase activity in the presence of 0.5 µmol/L dexamethasone (Dex) by the normalized luciferase activity produced in unstimulated transfectants. Mutation of GRE I abolishes the approximately 30-fold induction of the luciferase reporter. Mutation of GRE II attenuates but does not abolish the glucocorticoid effect. Reproduced with permission from Brasier et al.26 C, Schematic of mechanism of glucocorticoid-inducible regulation of AGT promoter. Glucocorticoid hormones enter the hepatocyte cytoplasm via facilitated diffusion. Upon binding to the high-affinity 8S steroid receptor complex, the GR disassociates from heat-shock protein 90 (HSP 90) (reviewed in Reference 53). The activated 4S form of the GR is translocated into the nucleus. Within the nucleus, the GR recognizes specific GREs and stimulates transcription of the AGT promoter.

A mechanism for GR induction of the AGT promoter is schematically presented in Fig 2C. The GR is a ligand-inducible transcription factor maintained in an 8S-heteromeric complex within the cytoplasm through interactions with heat-shock proteins -70, 90, and 56 (reviewed in References 53 and 55). Glucocorticoids, after entering the cell, bind to the receptor with nanomolar affinity. Upon binding to ligand, the 8S heteromeric GR complex disassociates into a 4S form that enters the nucleus. The transformed 4S receptor is now competent for binding to high-affinity binding sites within the genome. Once bound, GRs modulate gene expression through changes in chromatin structure (eg, disruption of nucleosome spacing) and recruitment of downstream factors.^{53 56 57} Whether GR interaction with the AGT promoter alters the chromatin structure or facilitates the binding of downstream activators is unknown presently and will require more detailed study.

	Mechanisms of AGT Gene Activation by Inflammation

Top Abstract Introduction Mechanisms of AGT Gene... Mechanisms of AGT Gene... Transcriptional Mechanism of the... Summary References

 
One well-characterized physiological activator of AGT expression is the hepatic APR, a highly species-conserved stereotypic response of the mammalian liver to the initiation of inflammation. Inflammation-stimulated AGT expression has been most clearly documented in experimental animals.^{31 34 35} The fact that plasma AGT levels are increased in human subjects with systemic infection⁵⁸ is consistent with this phenomenon being applicable to regulation of the human gene, but the few reported studies on the effect of inflammation on the human gene preclude definitive conclusions. In the APR, local injury or inflammation results in cytokine elaboration; these hormones induce a switch in hepatic gene transcription to producing proteins involved in macrophage opsonization and wound repair.⁴ The APR, initiated experimentally by intraperitoneal administration of bacterial lipopolysaccharide, is a potent inducer of hepatic AGT expression.^{31 34 35} Initiation of systemic inflammation by a single dose of intraperitoneal lipopolysaccharide results in a fivefold increase in hepatic steady-state mRNA levels at 3 hours of injection³¹ and a threefold increase in plasma AGT at 8 hours.⁵⁹ In the lipopolysaccharide-induced APR, the macrophage-derived cytokines TNF- and interleukin-1 are likely to be the key activators of hepatic AGT expression, although in some in vitro conditions, interleukin-6 can activate AGT expression.⁶⁰ Surprisingly, another model of inflammation, initiated by turpentine,³⁵ does not uniformly induce AGT gene expression, indicating differences in the cytokine response to different nonspecific inducers of inflammation.

Study of AGT during the APR has provided important insights into transcriptional control elements and DNA binding proteins that control expression of the rat AGT gene (reviewed in References 30 and 61). One crucial DNA control element of the AGT promoter, located between -557 and -531 in the rat gene (5' to the transcriptional start site) is absolutely required for cytokine induction of AGT promoter activity. This cytokine-inducible region, containing the dyad-symmetric sequence 5'-GTTGGGATTTCCCAAC-3', was termed the APRE. Site-directed mutations of the rat AGT promoter within this element completely block cytokine inducibility in stable hepatocyte transfectants and transiently transfected promoters.^{31 62} Moreover, the APRE is a TNF-–inducible enhancer that confers TNF- induction onto an inert minimal promoter, indicating that it functions as a bona fide cytokine-inducible enhancer.^{31 62}

Enter NF-B
The APRE functions as a cytokine-inducible enhancer because it is a binding site for the potent NF-B transcription factor complex. NF-B is a multiprotein complex encoded by different genes, with each product 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 63). Rel A is a powerful transactivating NF-B family member by virtue of its unique COOH-terminal transactivating domain.^{64 65} By contrast, 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.^{66 67} Rel A/NF-B1 heterodimers have slightly different DNA binding preferences than either protein alone, and in the appropriate cell line they are inactivated by association with inhibitory cytoplasmic proteins.

In resting hepatocytes, NF-B is sequestered in the cytoplasm through reversible interactions with a family of inhibitory proteins termed IB.^{68 69 70} One IB protein identified in rat liver is the homologue of human MAD3 (IB).⁷¹ IB binds to NF-B1/Rel A through repetitive, conserved domains homologous to erythrocyte ankyrin. In so doing, IB prevents DNA binding activity of Rel A and masks its nuclear localization signal.⁷² Cytokines, such as TNF-, activate NF-B by disrupting its association with the IB inhibitor through a coupled phosphorylation-IB degradation step,^{67 73 74 75} 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.^{31 62} That the inert NF-B1 DNA binding subunit binds to the AGT APRE in unstimulated hepatocyte nuclei has been shown by cross-competition using NFB1 binding site oligonucleotides for in vitro DNA binding assays (Fig 3)^{26 32} and inclusion of NF-B1 subunit–specific antibodies in gel mobility "supershifting" assays (Fig 4).⁶² All of these studies demonstrate that the DNA binding activity of NF-B1 is present in the nuclei of unstimulated cells where APRE transcription is low.⁶² After TNF- treatment, however, the amount of NF-B1 bound by the APRE increases, but the transcriptionally potent Rel A, previously undetectable, becomes a major APRE binding protein (Figs 3 and 4). The induction of APRE transcription occurs in parallel with the appearance of the NF-B1/Rel A nucleoprotein complex.⁶² Taken together, these data can be interpreted as indicating that Rel A is the relevant APRE transactivator in response to TNF- treatment.

View larger version (89K): [in this window] [in a new window]   Figure 3. TNF- induces NF-B proteins to bind the AGT APRE in a sequence-specific fashion. Nuclear extracts from unstimulated or TNF-–stimulated HepG2 cells were used in gel mobility shift assays with or without unlabeled APRE DNA binding competitor DNA. Shown is the autoradiogram using radiolabeled APRE wild-type (WT) DNA. Both C1 and C2 are completely competed with a 100-fold excess of unlabeled APRE WT DNA but not by APRE M6 or APRE M2 DNA binding sites (see Fig 2A for sequences). These observations indicate that the constitutive C2 complex and the TNF-–inducible C1 complex both bind DNA in a sequence-specific manner. Reproduced with permission from Brasier et al.62

View larger version (70K): [in this window] [in a new window]   Figure 4. Gel mobility "supershift" assay demonstrates that NF-B subunits NF-B1 and Rel A are within the APRE nucleoprotein complex. Nuclear extracts from unstimulated or TNF-–stimulated HepG2 cells were used in gel mobility "supershift" assays with radiolabeled APRE DNA in the presence of subunit-specific NF-B antibodies against NF-B1 (p50), NF-B2 (p49), c-Rel (Rel B), or Rel A (p65). This assay will produce an additional more slowly moving "supershift" band composed of antibody/NF-B subunit/DNA complex. We used a 5% polyacrylamide gel to visualize the supershifted bands; the free probe was electrophoresed off the gel. As with preimmune serum (PI), the addition of anti–NF-B2 or c-Rel antibodies does not perturb the nucleoprotein complex. Addition of anti–NF-B1 antibodies produces an additional complex in both 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 supershifted 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. Reproduced with permission from Brasier et al.62

Analysis of transiently expressed mutants of the Rel A transactivator (expressed as a fusion protein with the yeast GAL4 DNA binding domain) in HepG2 hepatocytes has allowed the identification of a domain necessary for TNF- induction (summarized in Fig 5A). Deletion of amino acids 1-254 blocks TNF- induction of GAL4/Rel A 255-551 but does not inhibit its function as a transactivator.⁶² These observations indicate the requirement of amino acids 1-254 for cytokine induction. This domain is involved in DNA binding, interaction with IB, nuclear localization, and dimerization and also contains protein kinase A and PKC phosphoacceptor sites.^{65 76 77} 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 interaction with IB. The deletion of the Rel homology domain prevents sequestration of Rel A in the cytoplasm, allowing Rel A to enter the nucleus and stimulate reporter gene activity independently of the TNF- hormone.

View larger version (18K): [in this window] [in a new window]   Figure 5. Domain organization of APRE binding proteins. A, NF-B family. NF-B1 and Rel A, encoded by distinct genes, share a common approximately 250–amino acid NH2-terminal Rel homology domain. Within this region lies the DNA binding domain, dimerization domain, and IB-interaction domains. NF-B1 is processed from a larger precursor containing at its COOH-terminus ankyrin repeats (processing site is indicated by arrows). In Rel A, the unique COOH-terminus contains two transcriptional activation domains.63 64 65 NF-B1 lacks this region and so is an inert DNA binding protein. The hormone-inducible region of Rel A is identified.62 NLS indicates nuclear localization signals. B, C/EBP transcription factor family. C/EBP and NF-IL6 represent the major C/EBP family members that bind the AGT APRE.32 79 These proteins share a common approximately 80–amino acid COOH-terminal basic domain/leucine zipper motif that contains the DNA contact and dimerization domains.79 80 The transactivation domains of the C/EBPs are rich in alanine and proline residues, termed trans-activation elements (TE) I to TE III.81 NF-IL6 contains an N-terminal activation domain and cell-specific repression domains. In addition, NF-IL6 contains serine and threonine phosphoacceptor sites that are targets for activated protein kinase A (RKSRDK),82 mitogen-activated kinase (PGTPSPA) activated by the ras proto-oncogene,83 and within the COOH-terminal leucine zipper region, a calcium/calmodulin–dependent kinase site (RELST).84 Phosphorylation of these residues stimulates the activity of the NF-IL6 transactivator (phosphoacceptor sites are indicated by vertical arrows). CSSD indicates complex stabilizing subdomain. C, Schematic of mechanisms of AGT gene activation by the APR. Cytokines interleukin-1 (IL-1) and TNF- are elaborated by activated macrophages at the site of injury. These hormones bind to specific cell surface receptors on the hepatocyte and initiate second messenger signaling events.93 NF-B is sequestered in the cytoplasm by association with the IB inhibitor. Within minutes of TNF- stimulation, the Rel A/NF-B1 heterodimer is released from the IB inhibitor and translocates into the nucleus. Rel A/NF-B1 binds and activates the APRE. At a slower temporal rate (hours), the nuclear C/EBP is degraded, followed by its replacement by the NF-IL6 transcription factor.

Recruitment of Rel A, the NF-B subunit with potent transactivation properties, into the APRE/NF-B1 complex is thus vital to the trans-activation process. Determination of the molecular mechanisms by which Rel A activates the AGT promoter will require additional study. NF-B1/Rel A stimulates the transcription of the human immunodeficiency virus promoter in a fashion dependent on the addition of cofactor fractions as well as the basal transcription factors.⁷⁸ Whether AGT gene activation by NF-B1/Rel A requires similar cofactors will require further study. In addition, after DNA binding, Rel A bends DNA; this property may indicate that one mechanism that Rel A uses to activate AGT gene expression is through "looping," where the APRE could be brought into close contact with the basal transcription machinery.

C/EBP Exchange During the APR
Analysis of APRE binding proteins isolated from rat hepatocytes led to the identification of a family of heat-stable DNA binding proteins that also contact the APRE in a sequence-specific fashion (summarized in Fig 5B). These proteins, members of the C/EBP family, interact with guanosine residues distinct from and overlapping with those bound by the NF-B complex.³² C/EBP is constitutively expressed in hepatocytes. By contrast, another C/EBP family member, NF-IL6, is itself an acute-phase–inducible protein in hepatocytes in which its abundance increases in response to inflammation.⁸⁰ NF-IL6 is the human homologue of rat liver–enriched transcriptional activator protein,⁸⁵ interleukin-6 DNA binding protein,⁸⁶ ₁ acid glycoprotein enhancer binding protein,⁸⁷ mouse C/EBPß,⁸⁸ and C/EBP-related protein 2.⁸⁹ Within the NH₂-terminus are stretches of alanine and proline residues that function in cell type–specific promoter activation.⁹⁰ Moreover, NFIL6 is a protein whose activity can be modulated by protein kinase A,⁸² mitogen-activated kinase,⁸³ and calcium-calmodulin–dependent kinase⁸⁴ and related phosphatases by phosphorylation/dephosphorylation in the appropriate cell type.

At the COOH terminus of the protein lies its DNA binding domain, through which NF-IL6 recognizes the APRE by an 81-residue basic region/leucine zipper motif. We described the use of controlled tryptic protease digestion of recombinant NF-IL6/APRE complex to identify the core DNA binding domain.⁷⁹ Within the basic region/leucine zipper DNA binding domain lies a stretch of six amino acids whose deletion markedly destabilizes the DNA complex, termed the complex stabilizing subdomain.⁷⁹

The precise role of the C/EBPs in regulating AGT expression has remained elusive, particularly because of the lack of suitable in vitro models for their study. In vitro DNA binding assays demonstrate that the NF-B1/Rel A protein and C/EBPs bind to the APRE in a mutually exclusive manner.^{32 61} In unstimulated hepatocytes, in which the C/EBPs are less potent transactivators than Rel A, C/EBP members function as competitive inhibitors of Rel A activity by virtue of displacing the more potent transactivator. Nevertheless, this observation is of uncertain relevance to the biological situation of the hepatic APR because potential signals that modify NF-IL6 activity may not be present. Several clues indicate the potential importance of the C/EBPs for AGT expression. First, the C/EBPs are expressed in high abundance in both hepatocytes and adipocytes. On a weight basis, both of these tissues produce the highest levels of AGT mRNA. Second, that C/EBP, the most potent APRE transactivator of the C/EBP family, is first expressed in rodent liver immediately postpartum⁹¹ at a time when AGT synthesis can first be detected²⁰ perhaps suggests its role in constitutive AGT gene expression during development. Moreover, in hepatocytes and adipocytes, cytokine stimulation produces a fall in the constitutive C/EBP and a slower increase in NF-IL6 protein.^{23 92} During this exchange of C/EBPs, a "window" may be opened for the NF-B transcription factor to access the APRE during the APR.

In summary, circulating cytokines secreted from activated macrophages are effectors of the highly conserved APR in vertebrates. The cytokines interleukin-1 and TNF- bind to specific hepatocyte receptors, activating second messenger signaling cascades that ultimately control the nuclear expression of the two transcription factor families that bind to the APRE. Rel A/NF-B1 is a transcription factor complex that, like the GR, is a preformed complex in the cytoplasm associated with inhibitory proteins (IB). Rel A/NF-B1 is released from the inhibitor in response to cytokine signaling events, allowing Rel A/NF-B1 to translocate into the nucleus and stimulate transcription. A schematic model for the mechanism of AGT gene expression during the APR is presented in Fig 5C.

	Transcriptional Mechanism of the RAS Positive Feedback Loop

Top Abstract Introduction Mechanisms of AGT Gene... Mechanisms of AGT Gene... Transcriptional Mechanism of the... Summary References

 
Intravascular Ang II formation controls the activity of the RAS through negative feedback regulation of renin secretion and positive feedback regulation of AGT synthesis.^{37 38 39} This control is important in renovascular hypertension and bilateral nephrectomy, in which AGT transcripts accumulate in the liver.^{36 39} In the hepatocyte, Ang II stimulates AGT gene expression in part through enhanced transcription, as documented by nuclear run-on analysis.³⁷ The RAS "positive feedback loop" is important to ensure that sufficient AGT is in supply to respond to any future hypotensive challenges. Moreover, the positive feedback loop may also play a pathophysiological role in the malignant phase of essential or renovascular hypertension, in which enhanced AGT synthesis initially sustains the elevated blood pressure,^{6 36 38 39 94} although this point is controversial.⁹⁵ Understanding the mechanisms for the RAS positive feedback loop will therefore elucidate the pathophysiology of these disease states.

The identification of the cis-regulatory element(s) and corresponding trans-acting factor(s) mediating the positive feedback loop on AGT gene synthesis has remained elusive. One difficulty in studying Ang II–dependent enhancers has been the loss of normally expressed AT₁ receptor expression after isolation of primary hepatocytes and the lack of AT₁ expression in transformed hepatocyte cultures.⁹⁶ The molecular cloning of AT₁ has led to the development of an important reagent to address the mechanism of the RAS positive feedback loop.⁹⁷ Transient high-level expression of AT₁ results in high-affinity Ang II binding activity that is functionally coupled to intracellular calcium transients.⁹⁷ Using transcription assays in AT₁-complemented human hepatocytes, we have shown that the AGT multihormonal response element, spanning nucleotides -615 to -470, is an Ang II–inducible enhancer.⁹⁶ Transcription driven by the multihormone-inducible enhancer occurs over a physiological dose-responsive range producing a statistically significant threefold induction that peaks at a concentration of 10 nmol/L Ang II (identical to the K_d of AT₁).⁹⁷ Mutations of the multihormone-inducible enhancer that blocks NF-B binding also abolished Ang II induction of the transfected AGT transgenes.⁹⁶ The APRE is an Ang II–inducible enhancer because it confers Ang II inducibility onto an inert promoter driving the luciferase reporter (Fig 6 and Reference 96). The Ang II–inducible APRE activity was first detectable at 0.01 nmol/L Ang II, with the maximal 13-fold induction seen in response to 10 nmol/L Ang II. Higher doses of up to 100 nmol/L gave less activity than the maximum 13-fold, perhaps because of the well-described phenomenon of agonist-induced receptor downregulation.⁹⁹ Importantly, the fact that the Ang II–dependent transcriptional activation was seen only in cells cotransfected with the AT₁ expression vector indicates that AT₁ is absolutely required for this effect.⁹⁶ As with the site-directed mutations within the native AGT multihormone-inducible enhancer, Ang II stimulation of the APRE depends on NF-B binding because no transcriptional induction by Ang II was seen in either APRE M6 (a mutant that binds C/EBP only) or APRE M2 (a mutant that binds neither NF-B nor C/EBP) driving the luciferase reporter gene in response to Ang II.

View larger version (22K): [in this window] [in a new window]   Figure 6. Ang II and TNF- stimulate APRE transcription through pharmacologically distinct pathways. A, APRE-linked luciferase reporter; B, effect of staurosporine on Ang II (AII in figure) and TNF- induction of APRE-driven luciferase reporter activity. HepG2 cells were transiently transfected with three copies of wild-type APRE 5' of the AGT promoter–driven luciferase reporter gene (A). One hour before hormone stimulation, vehicle (dimethyl sulfoxide) or staurosporine (in dimethyl sulfoxide) was added to a final concentration of 50 nmol/L.98 Left, Mean±SD (n=3 experiments) of normalized luciferase reporter activity after stimulation with increasing doses of TNF-. APRE transcription is stimulated 25-fold by 30 ng/mL TNF-; the presence of staurosporine does not significantly alter the induction. Right, Mean±SD (n=5 experiments) of normalized luciferase reporter activity after stimulation with increasing concentrations of Ang II. Ang II stimulates APRE transcription 11-fold at 1 and 10 nmol/L; in the presence of staurosporine, a maximum of 2.8-fold induction is produced. Compared with vehicle, staurosporine inhibition is statistically significant at 0.1, 1.0, and 10 nmol/L Ang II (P<.05). C, Effects of PKC downregulation on Ang II and TNF- induction of APRE transcriptional activity. Transiently transfected HepG2 cells were exposed to either vehicle alone (open bars) or 0.5 µmol/L phorbol 12-myristate 13-acetate (PMA) (solid bars) for 20 hours before a 3-hour washout to allow recovery of TNF- receptor expression.98 Cells were then stimulated for 4 hours before harvest and assay for luciferase. Left, PKC downregulation of TNF- induction. Group 1, unstimulated controls; group 2, acute stimulation with 0.5 µmol/L PMA; group 3, stimulation with 3 ng/mL TNF-; group 4, stimulation with 30 ng/mL TNF-. Acute (4-hour) stimulation with PMA produces a 25-fold induction of APRE-luciferase reporter activity that is completely blocked by the chronic PMA exposure, indicating hydrolysis of PKC activity. After PKC downregulation, TNF- induction is decreased to 8-fold (3 ng/mL TNF-) and 12-fold (30 ng/mL TNF-). Right, PKC downregulation of Ang II induction. Group 1, unstimulated controls; group 2, acute stimulation with 0.5 µmol/L PMA; group 3, stimulation with 1 nmol/L Ang II; group 4, stimulation with 10 nmol/L Ang II. As in the left panel, after acute stimulation with PMA, the 25-fold induction of APRE-luciferase reporter activity is completely blocked by the chronic PMA exposure, indicating PKC hydrolysis. That there is no significant induction of reporter activity by Ang II after chronic PMA exposure indicates that PKC is essential for Ang II activation of APRE transcription.

Continuously perfused primary hepatocytes have been used to demonstrate that Ang II induces AGT mRNA and protein.⁴⁰ With the gel mobility shift assay, a fourfold increase of APRE binding activity by Ang II was observed in a dose-dependent fashion. First detectable at 0.5 nmol/L Ang II, APRE binding activity peaks at a physiological concentration of 5 nmol/L Ang II. Continuous infusion of 50 nmol/L Ang II produced no greater stimulation, so the effect of Ang II is saturable. This concentration range is indistinguishable from that required for transcriptional activity in transient HepG2 transfectants. Moreover, the increase in APRE DNA binding activity was rapid and observed as early as 1 hour after stimulation. Competition analysis showed that Ang II–activated APRE binding is sequence specific for the NF-B protein family. The Rel A transcriptional activator was identified in this complex with subunit-specific antibodies in the gel mobility "supershift" assay.

Ang II– and TNF-–Induced Signals Are Convergent on NF-B Rel A
The finding that Ang II and TNF- activate Rel A indicates that in some cells (such as hepatocytes), postreceptor cytokine and cardioactive peptide signaling pathways are convergent (Fig 7). In a wide variety of cell types, TNF- activates NF-B through two mechanisms: (1) a phosphatidylcholine-specific phospholipase C–coupled activation of an acidic sphingomyelinase with formation of ceramide as a second messenger, and (2) 1,2-diacylglycerol formation resulting from PKC activation.^{98 99 100 101} Either of these pathways appears to be sufficient to induce the proteolysis of IB through a coupled phosphorylation/degradation pathway.

View larger version (24K): [in this window] [in a new window]   Figure 7. A model for the regulation of rat AGT multihormone-inducible enhancer (nucleotides -615 to -470). The AGT multihormone-inducible enhancer is a recognition sequence for distinct classes of inducible transcription factors.26 32 51 62 96 Liganded GR binds to GREs 1 and 2. Cytokines induce the cytoplasmic-nuclear translocation of the NF-B complex, composed primarily of the NF-B1/Rel A subunits, and binds to the APRE. Ang II (AII in the figure), via the AT1 receptor, also converges on the NFB1/Rel A transcription factor complex, allowing for cytokine and Ang II "cross talk." AT1 receptor is coupled to the NF-B/IB complex through a staurosporine-sensitive pathway and also depends on PKC. IL-1 indicates interleukin-1; Stauro, staurosporine; and HSP, heat-shock protein.

In contrast, on binding to the hepatocyte type 1 receptor, Ang II stimulates the formation of 1,2-diacylglycerol with consequent PKC activation, mobilization of intracellular calcium, and inhibition of hormone-stimulated adenylate cyclase activity.^{97 99 102 103} Of these, PKC appears to be an important molecule for Ang II to induce early gene (c-fos) synthesis. Chronic exposure to phorbol agonist (to downregulate PKC) completely inhibits c-fos induction in cardiomyocytes¹⁰⁴ and partly antagonizes c-fos induction in glomerulosa cells¹⁰⁵ or vascular smooth muscle cells.¹⁰⁶ Although the relevant signaling events activated by Ang II that control NF-B activity remain speculative, the demonstration that certain PKC isoforms are physically associated with the IB molecule (kinases that can phosphorylate and inactivate this inhibitor in vitro¹⁰⁷ ) makes PKC a likely candidate for coupling the activated AT₁ receptor to modulating NF-B activity.

We note the recently reported observations in vascular smooth muscle cells that AT₁ receptor is functionally coupled to a distinct cytoplasmic-inducible transcription factor family (the Jak/STAT pathway).¹⁰⁸ Although this pathway has not been implicated in AGT regulation, these observations provide additional pathways for cytokine and Ang II intracellular "cross talk" linked through the activated Ang II receptor.

Ang II and TNF- Activate Rel A Through Pharmacologically Distinct Intracellular Signaling Pathways
Although the rapid induction of Rel A DNA binding and transcriptional activity by Ang II is similar to the kinetics of Rel A activation by TNF- reported in our earlier studies,⁶² we consistently observed that Ang II was a less-potent APRE stimulus than TNF-, perhaps indicating that these agents were acting through different intracellular messengers. We therefore used transient transfection assays to determine whether Ang II and TNF- were activating APRE transcription via similar or distinct intracellular signals. Both Ang II and TNF- induction of APRE-luciferase reporter activity are critically dependent on the activity of the phosphatidylcholine-specific phospholipase C because the specific phosphatidylcholine-specific phospholipase C inhibitor tricyclodecan-9-yl xanthogenate (D609) completely blocks reporter induction by either hormone (data not shown).^{100 104} In contrast, the induction by Ang II of luciferase reporter activity was completely blocked by the protein kinase inhibitor staurosporine (50 nmol/L), and TNF- induction was unaffected at the same inhibitor concentrations (Fig 6B). In addition, chronic agonist downregulation of PKC activity also completely blocked the effect of Ang II but only partially affected the activity of TNF- on APRE-luciferase reporter activity (Fig 6C). Taken together, these data indicate the existence of pharmacologically distinct second messenger pathways by which Ang II and TNF- activate APRE transcription.

	Summary

Top Abstract Introduction Mechanisms of AGT Gene... Mechanisms of AGT Gene... Transcriptional Mechanism of the... Summary References

 
In this article we have reviewed the transcriptional mechanisms used by potent physiological inducers of hepatic AGT transcriptional activity. It is important to recognize that in other tissues expressing local RASs, AGT synthesis may be regulated through other mechanisms. That this may be the case is underscored by the tissue-specific differences in AGT induction by the APR, where lipopolysaccharide increases AGT mRNA in all tissues except the brain, and by the distinct temporal profile of Ang II–stimulated AGT synthesis in the liver compared with cardiac myocytes. Our studies, using the hepatocyte as a model, indicate that AGT synthesis is tightly controlled through the actions of a multihormone-inducible enhancer, located in the rat gene between -615 and -470 nucleotides upstream of the transcriptional start site. Within this element are binding sites for distinct classes of inducible transcription factors. Glucocorticoid hormones induce AGT transcription by binding directly to the GR. The GR is translocated into the nucleus to interact with discrete and hierarchical GREs within the AGT promoter. Cytokines (TNF- and interleukin-1) and the vasoactive Ang II peptide produce AGT transcription through a distinct cytoplasmically inducible transcription factor composed of the NF-B subunit NF-B1/Rel A. NF-B1, the inert DNA binding subunit, is found in nuclei from unstimulated cells. Recruitment of the potent transcriptionally active Rel A subunit is necessary for AGT promoter induction. This process occurs through a signal-induced phosphorylation/IB degradation step, resulting in nuclear translocation of the Rel A DNA binding activity. Finally, our present data indicate that the cytokine TNF- and the vasopressor Ang II converge on Rel A activator but do so through a pharmacologically distinct pathway (Fig 7). These studies link the intracellular actions of cytokines and Ang II.

	Selected Abbreviations and Acronyms

 

AGT = angiotensinogen Ang I, II = angiotensin I, II APR = acute-phase response APRE = acute-phase response element AT1 = angiotensin type 1 C/EBP = CCAAT/enhancer binding protein GR = glucocorticoid receptor GRE = glucocorticoid response element NF-IL6 = nuclear factor–interleukin-6 NF-B = nuclear factor-B PKC = protein kinase C RAS = renin-angiotensin system Rel A = nuclear factor-B p65 subunit TNF- = tumor necrosis factor-

	Acknowledgments

 
This work was supported in part by National Institutes of Health grant 1 R29 HL-45500. A.R.B. is an Established Investigator of the American Heart Association. We thank David Konkel for review of the manuscript and helpful suggestions.

	References

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