Angiotensinogen Promoter Research Articles

Identification of two distinct Sp1- and RBF-1-like nuclear factors that bind to the upstream region of the human angiotensinogen promoter

Angiotensionogen, the protein precursor of angiotensin II that is a crucial regulator of blood pressure and electrolyte balance, is constitutively produced by the liver. In the present study, we identified two nuclear factors that are possibly involved in maintaining the constitutive promoter activity of the human angiotensinogen gene. The 32 bp DNA region between -344 and -313 located in the 1.3 kb angiotensinogen upstream region (-1222 to +44) partially contributed to the maintenance of the efficient promoter activity in HepG2 cells. This segment was able to form the complexes with HepG2 nuclear extracts, which could be dissociated by competing recognition sequences that contain those of either Sp1 or RBF-1. Anin vivo competition experiment demonstrated that the parental promoter activity is reduced about 65% by an RBF-1 competitor more effectively than by an Sp1 competitor. These results suggested that Sp1- and RBF-1-like factors play roles in maintaining the constitutively active angiotensinogen promoter.

Angiotensinogen Gene Expression Is Induced by Cyclical Mechanical Stretch in Cultured Rat Cardiomyocytes

The effect of cyclical mechanical stretch on angiotensinogen gene expression was examined using a neonatal rat cardiocyte culture system. Cultured cardiocytes grown on a flexible membrane base were stretched by vacuum to 20% of maximum elongation, at 60 cycles/min. The angiotensinogen gene was activated 2 to 5 fold after stretch for 3 to 24 hr, as shown by quantitative reverse transcription polymerase chain reaction. The 5′-flanking region of the angiotensinogen promoter was activated after stretch for 24 hr. This gene expression could be completely suppressed by losartan, a specific antagonist of angiotensin II receptor. These results indicate that (1) cyclical mechanical stretching of cardiocytes is a good model for the study of cardiac hypertrophy-related gene expression; (2) cyclical stretch up-regulates expression of the angiotensinogen gene and (3) the increase in promoter activity may contribute to the induction of angiotensinogen mRNA by cyclical stretch.

Neuroectodermal tumors expressing c-, L-, and N-myc in transgenic mice that carry the E1A/E1B gene of human adenovirus type 12.

Adenovirus early 1 (E1) region gene products, including E1A and E1B, are required for transformation of primary cultured rodent cells. In order to investigate in vivo action of the E1 region, we established a line of transgenic mice carrying the oncogenic E1A and E1B genes of human adenovirus type 12 under control of the human angiotensinogen promoter. Histopathological analyses indicated that transgenic mice heritably develop neuroectodermal tumors arising from the pelvic region with varying degrees of incidence. The transgene was expressed in the neuroectodermal tumors as well as in TNT-1 cells, a cell line established from the tumors, where the human angiotensinogen promoter was constitutively active. The high level expression of c-, L-, and N-myc without gene amplification was notable in the original tumors and TNT-1 cells, but not in another tissue examined. The co-expression of the three sets of myc family genes in both the original tumors and the established cell line provided the possibility that the target cells for transformation may belong to a specific cell type that expresses all these oncogenes during development.

Identification of cell type-dependent enhancer core element located in the 3'-downstream region of the human angiotensinogen gene.

We previously demonstrated that the 3.8-kilobase DNA fragment containing exons 4 and 5 of the human angiotensinogen (hAG) gene enhances the expression of chloramphenicol acetyltransferase gene, under control of the hAG promoter, in human hepatoma HepG2 cells. In the present study, to define regulatory elements of the hAG gene, we have functionally dissected this downstream region and localized a cell type-dependent enhancer to the 832-base pair sequence containing the exon 5 and 3'-flanking region. Further deletion analyses revealed that the 24-base pair core DNA fragment present in the 3'-flanking region was responsible for this enhancement. Electrophoretic mobility shift assay demonstrated that the 3'-enhancer core element interacts specifically with two nuclear factors from the HepG2 cells, one of which is an uncharacterized factor (human angiotensinogen enhancer factor-1: hAEF-1), the other is an AP-3-related factor. Mutation analyses indicated that the disruption of hAEF-1 binding alone completely impaired the enhancer activity of the core element. These results suggested that the downstream enhancer core element interacting with hAEF-1 plays an important role in activating the angiotensinogen promoter in a cell type-dependent manner.

Molecular mechanism of transcriptional activation of angiotensinogen gene by proximal promoter.

Angiotensinogen is shown to be produced by the liver and the hepatoma cell line HepG2. As a first step for understanding the molecular relationship between the transcriptional regulation of the angiotensinogen gene and the pathogenesis of hypertension, we have analyzed the basal promoter of the angiotensinogen gene. Chloramphenicol acetyltransferase (CAT) assays with 5'-deleted constructs showed that the proximal promoter region from -96 to +22 of the transcriptional start site was enough to express HepG2-specific CAT activity. Electrophoretic mobility shift assay and DNase I footprinting demonstrated that the liver- and HepG2-specific nuclear factor (angiotensinogen gene-activating factor [AGF2]) and ubiquitous nuclear factor (AGF3) bound to the proximal promoter element from -96 to -52 (angiotensinogen gene-activating element [AGE2]) and to the core promoter element from -6 to +22 (AGE3), respectively. The site-directed disruption of either AGE2 or AGE3 decreased CAT expression, and the sequential titration of AGF3 binding by in vivo competition remarkably suppressed HepG2-specific CAT activity. Finally, the heterologous thymidine kinase promoter assay showed that AGE2 and AGE3 synergistically conferred HepG2-specific CAT expression. These results suggest that the synergistic interplay between AGF2 and AGF3 is important for the angiotensinogen promoter activation.

Molecular mechanism of adipogenic activation of the angiotensinogen gene.

Angiotensinogen gene expression is controlled in a tissue- and development-specific manner. Interestingly, the angiotensinogen gene is abundantly expressed in adipose tissues other than the liver, where it is mainly produced. We investigated the molecular mechanism of angiotensinogen gene expression in a 3T3-L1 preadipocyte-adipocyte system. Although angiotensinogen mRNA was barely detectable in preadipocytes, its levels increased significantly during differentiation. As a whole, the pattern of the change in transcriptional activity of the angiotensinogen promoter was similar to that of the angiotensinogen mRNA levels during adipogenic differentiation, indicating that the activation of the angiotensinogen promoter might be involved in the adipogenic differentiation-coupled gene expression. The proximal promoter region, from -96 to +22 of the transcriptional start site, was sufficient to confer adipogenic activation, and the proximal element from -96 to -52 of the transcriptional start site was necessary for this promoter stimulation. DNA-protein binding experiments showed that this proximal element specifically bound to a nuclear factor induced by adipogenic differentiation. These results suggest that the proximal promoter element from -96 to -52 plays a role in adipogenic activation of the angiotensinogen promoter.

Proximal and core DNA elements are required for efficient angiotensinogen promoter activation during adipogenic differentiation

Angiotensinogen is abundantly expressed in adipose tissue as well as in liver where it is mainly produced. To address the mechanism of this adipogenic expression, promoter regions of the mouse angiotensinogen gene are fused to the chloramphenicol acetyltransferase reporter gene and stably transfected into 3T3-L1 preadipocytes. Promoter activity correlates well with an increase of mRNA levels during adipogenic differentiation, thereby demonstrating that the induction is primarily due to transcriptional activation. Deletion analysis indicates that the proximal promoter region from -96 to +22 is able to mediate the chloramphenicol acetyltransferase induction and identifies two transcriptionally active regions: AGE1 (position -399 to -139) and AGE2 (position -96 to -52). Heterologous promoter assay reveals that AGE1 behaves with a constitutive enhancer-like property and that AGE2 functions as a differentiation-inducible activator. Gel shift experiments show that AGE2 specifically binds a novel factor (AGF2), which is induced upon differentiation. Furthermore, a constitutive factor (AGF3) binds to the core promoter region including the exon 1 (from -6 to +22, AGE3). Mutations within either AGE2 or AGE3 that disrupt nuclear factors binding in vitro dramatically reduced the chloramphenicol acetyltransferase activation in the native promoter context. These results suggest that both AGE2 and AGE3 are necessary for mediating efficient activation of the mouse angiotensinogen promoter during adipogenic differentiation.

Transcriptional regulation of hepatic angiotensinogen gene expression by the acute-phase response

The acute-phase response is a protective physiological reaction to tissue injury manifested by the immediate increase in production and secretion of liver proteins the function of which is to re-establish the homeostasis altered by injury. Such proteins include blood coagulation factors, opsonins, protease-inhibitors and angiotensinogen, a precursor of the potent vasopressor peptide angiotensin II. The angiotensinogen gene is typical of genes regulated during the acute-phase response inasmuch as the promoter regulating its transcription rate is acutely responsive to three known mediators of the acute-phase response: glucocorticoids, and the cytokines interleukin-1 and tumor necrosis factor. We present a model, based on experimental evidence, for the mechanism by which angiotensinogen gene transcription is regulated in a graded fashion by the interplay of several hormonally-inducible transcription factors that bind a hormonally-inducible enhancer unit of the angiotensinogen promoter. These factors include the glucocorticoid receptor, nuclear factor kappa B and members of the CAAT/viral enhancer (C/EBP) family of DNA-binding proteins.

A family of constitutive C/EBP-like DNA binding proteins attenuate the IL-1 alpha induced, NF kappa B mediated trans-activation of the angiotensinogen gene acute-phase response element.

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 kappa B (NF kappa B). 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 forming 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. Assays of APRE-luciferase reporter plasmids transfected into HepG2 cells show that a mutated APRE that binds only BPi functions as an IL-1 alpha inducible enhancer, whereas a mutated APRE that binds only BPc does not. The APRE mutant that binds the C/EBP-like BPcs to the exclusion of BPi functions as an uninducible basal enhancer both in the native context of the angiotensinogen gene and when multimerized and placed upstream of a minimal angiotensinogen promoter. The wild-type APRE that binds both BPi and BPc is less inducible by IL-1 alpha than the mutated APRE that binds only BPi. Gel shift competition assays demonstrate in vitro that the mechanism of transcriptional regulation by the APRE involves a competition between BPc and the inducible BPi for binding to the APRE. IL-1 alpha stimulation of hepatocytes leads to nuclear translocation of the NF kappa B-like BPi which competes with the constitutive C/EBP-like BPcs for overlapping binding sites on the APRE and thereby replaces weak transcriptional activators with a stronger one.