Sudhir Jain

A single-nucleotide polymorphism in human angiotensinogen gene is associated with essential hypertension and affects glucocorticoid induced promoter activity.

Hypertension is a serious health problem particularly for African-Americans. Previous studies have suggested that angiotensinogen (AGT) gene locus is involved in human essential hypertension. We have recently shown that an A/G polymorphism at −217 in the promoter of the AGT gene is associated with essential hypertension especially in African-Americans. We report here that A/G polymorphism at −217 affects the glucocorticoid-induced promoter activity of the human AGT gene. We show that recombinant glucocorticoid receptor (GR) binds strongly to the AGT gene promoter when nucleoside A is present at −217, and dexamethasone treatment increases the interleukin 6 induced promoter activity of reporter constructs containing nucleoside A at −217. Similarly cotransfection of GR and C/EBPβ or C/EBPδ increases the promoter activity of reporter construct containing nucleoside A at −217. Since AGT is an acute phase protein, we propose that increased expression of −217A allele of the AGT gene by glucocorticoids and C/EBP family of transcription factors may be involved in essential hypertension.

A haplotype of the angiotensinogen gene is associated with hypertension in african americans.

1. Hypertension is a serious risk factor for myocardial infarction, heart failure, vascular disease, stroke and renal failure. The incidence of hypertension is 25–30% in the adult Caucasian population and complications due to hypertension are even greater in African Americans.

Transgenic Mice with −6A Haplotype of the Human Angiotensinogen Gene Have Increased Blood Pressure Compared with −6G Haplotype

Hypertension is a serious risk factor for cardiovascular disease, and the angiotensinogen (AGT) gene locus is associated with human essential hypertension. The human AGT (hAGT) gene has an A/G polymorphism at −6, and the −6A allele is associated with increased blood pressure. However, transgenic mice containing 1.2 kb of the promoter with −6A of the hAGT gene show neither increased plasma AGT level nor increased blood pressure compared with −6G. We have found that the hAGT gene has three additional SNPs (A/G at −1670, C/G at −1562, and T/G at −1561). Variants −1670A, −1562C, and −1561T almost always occur with −6A, and variants −1670G, −1562G, and −1561G almost always occur with −6G. Therefore, the hAGT gene may be subdivided into either −6A or −6G haplotypes. We show that these polymorphisms affect the binding of HNF-1α and glucocorticoid receptor to the promoter, and a reporter construct containing a 1.8-kb hAGT gene promoter with −6A haplotype has 4-fold increased glucocorticoid-induced promoter activity as compared with −6G haplotype. In order to understand the physiological significance of these haplotypes in an in vivo situation, we have generated double transgenic mice containing either the −6A or −6G haplotype of the hAGT gene and the human renin gene. Our ChIP assay shows that HNF-1α and glucocorticoid receptor have stronger affinity for the chromatin obtained from the liver of transgenic mice containing −6A haplotype. Our studies also show that transgenic mice containing −6A haplotype have increased plasma AGT level and increased blood pressure as compared with −6G haplotype. Our studies explain the molecular mechanism involved in association of the −6A allele of the hAGT gene with hypertension.

A haplotype of human angiotensinogen gene containing -217A increases blood pressure in transgenic mice compared with -217G.

The human angiotensinogen (hAGT) gene contains an A/G polymorphism at -217, and frequency of -217A allele is increased in African-American hypertensive patients. The hAGT gene has seven polymorphic sites in the 1.2-kb region of its promoter, and variant -217A almost always occurs with -532T, -793A, and -1074T, whereas variant -217G almost always occurs with -532C, -793G, and -1074G. Since allele -6A is the predominant allele in African-Americans, the AGT gene can be subdivided into two main haplotypes, -6A:-217A (AA) and -6A:-217G (AG). To understand the role of these haplotypes on hAGT gene expression and on blood pressure regulation in an in vivo situation, we have generated double transgenic mice containing human renin gene and either AA or AG haplotype of the hAGT gene using knock-in strategy at the hypoxanthine phosphoribosyltransferase locus. We show here that 1) hAGT mRNA level is increased in the liver by 60% and in the kidney by 40%; and 2) plasma AGT level is increased by approximately 40%, and plasma angiotensin II level is increased by approximately 50% in male double transgenic mice containing AA haplotype of the hAGT gene compared with the AG haplotype. In addition, systolic blood pressure is increased by 8 mmHg in transgenic mice containing the AA haplotype compared with the AG haplotype. This is the first report to show the effect of polymorphisms in the promoter of a human gene on its transcription in an in vivo situation that ultimately leads to an increase in blood pressure.

Dexamethasone Promotes Hypertension by Allele-specific Regulation of the Human Angiotensinogen Gene * □

Background: Glucocorticoids modulate the RAS and cause hypertension. Results: SNPs in the hAGT promoter form two haplotypes, −6A and −6G. Transgenic mice with haplotype −6A respond to dexamethasone with tissue-specific up-regulation of hAGT, increased plasma AngII, and hypertension. Conclusion: Haplotypes of the hAGT gene govern transcriptional response to dexamethasone. Significance: Polymorphisms in hAGT provide for genetic predisposition to glucocorticoid-induced hypertension. The human angiotensinogen (hAGT) gene has polymorphisms in its 2.5-kb promoter that form two haplotype (Hap) blocks: −6A/G (−1670A/G, −1562C/T, and −1561T/C) and −217A/G (−532T/C, −793A/G, −1074T/C, and −1178G/A). Hap −6A/−217A is associated with human hypertension, whereas Hap −6G/−217G reduces cardiovascular risk. Hap −6A/−217A has increased promoter activity with enhanced transcription factor binding, including to the glucocorticoid receptor (GR). Glucocorticoid therapy frequently causes hypertension, the mechanisms for which are incompletely understood. We have engineered double transgenic (TG) mice containing the human renin gene with either Hap of the hAGT gene and examined the physiological significance of glucocorticoid-mediated allele-specific regulation of the hAGT gene. We have also studied the consequential effects on the renin angiotensin system and blood pressure. TG mice with Hap −6A and −6G were treated with and without a low dose of a GR agonist, dexamethasone (2.5 μg/ml), for 72 h. We found greater chromatin-GR binding with increased GR agonist-induced hAGT expression in liver and renal tissues of Hap −6A mice. Additionally, dexamethasone treatment increased circulating hAGT and angiotensin II levels in Hap −6A mice, as compared with −6G mice. Importantly, GR agonist significantly increased blood pressure and redox markers in TG mice with Hap-6A of the hAGT gene. Taken together, our results show, for the first time, that glucocorticoids affect hAGT expression in a haplotype-dependent fashion with SNPs in Hap −6A favoring agonist-induced GR binding. This leads to increased expression of the hAGT, up-regulation of the renin angiotensin system, and increased blood pressure and oxidative stress in Hap −6A mice.

Variable Transcriptional Regulation of the Human Aldosterone Synthase Gene Causes Salt-Dependent High Blood Pressure in Transgenic Mice

Background—Aldosterone, synthesized in the adrenal cortex by the enzyme CYP11B2, induces positive sodium balance and predisposes to hypertension. Various investigators, using genomic DNA analyses, have linked -344T polymorphism in the human CYP11B2 (hCYP11B2) gene to human hypertension. hCYP11B2 gene promoter has 3 single-nucleotide polymorphisms in linkage disequilibrium: T/A at -663, T/C at -470, and C/T at -344. Variants ACT occur together and form the haplotype-I (Hap-I), whereas variants TTC constitute Hap-II. We hypothesize that these single-nucleotide polymorphisms, when present together, will lead to haplotype-dependent differences in the transcriptional regulation of the hCYP11B2 gene and affect blood pressure regulation. Methods and Results—We evaluated differences in tissue expression in vivo and consequential effects on blood pressure stemming from the 2 haplotypes. Novel transgenic mice with the hCYP11B2 gene, targeted to the mouse HPRT locus, with either Hap-II or Hap-I variant are used in this study. Our results show increased adrenal and renal expression of hCYP11B2 in transgenic mice with Hap-I when compared with mice with Hap-II. Importantly, we observed increased baseline blood pressure in Hap-I transgenic mice, an effect accentuated by a high-salt diet. Pathophysiological effects of elevated aldosterone were corroborated by our results showing upregulation of proinflammatory markers in renal tissues from the transgenic mice with Hap-I. Conclusions—These findings characterize the haplotype-dependent regulation of the hCYP11B2 gene where -344T serves as a reporter polymorphism and show that Hap-I leads to increased expression of hCYP11B2, with permissive effects on blood pressure and inflammatory milieu.

Metabolic Syndrome Induces Over Expression of the Human AT1R: A Haplotype-Dependent Effect With Implications on Cardio-Renal Function

BACKGROUND The transcriptional regulation of the human angiotensin receptor subtype 1 (AT1R) gene in pathophysiologies, like the metabolic syndrome, is poorly understood. The human AT1R gene has polymorphisms in its promoter that can be arranged in 2 haplotypes. Variants -810T, -713T, -214A, and -153A always occur together (Hap-I) and variants -810A, -713G, -214C, and -153G form Hap-II. We have hypothesized that high fat diet will alter cellular transcriptional milieu and increase hAT1R gene expression in a haplotype-dependent manner. This will set up an AT1R-mediated feed-forward loop promoting inflammation, oxidative stress, and hypertension in Hap-I mice. METHOD Since Hap-I of the human AT1R gene is associated with hypertension in Caucasians, we generated transgenic (TG) mice with Hap-I and Hap-II and studied the physiological significance of high fat diet (HFD) on haplotype specific gene expression. Animals were fed with HFD for 20 weeks followed by blood pressure (BP) analysis and collection of their tissues for molecular and biochemical studies. RESULTS After HFD treatment, as compared to Hap-II, TG mice with Hap-I show increased expression of hAT1R gene and higher BP; suppression of antioxidant defenses (HO1, SOD1) and increased expression of IL-6, TNFα, IL-1β, NOX1. In vivo ChIP assay has shown that transcription factors CEBPβ, STAT3, and USF bind more strongly to the chromatin obtained from Hap-I TG mice. CONCLUSIONS Taken together, our results suggest, that after HFD treatment, as compared to Hap-II, the TG mice with Hap-I overexpress the AT1R gene due to the stronger transcriptional activity, thus resulting in an increase in their BP.

Age-Related Expression of Human AT1R Variants and Associated Renal Dysfunction in Transgenic Mice

BACKGROUND The contribution of single nucleotide polymorphisms in transcriptional regulation of the human angiotensin receptor type I (hAT1R) gene in age-related chronic pathologies such as hypertension and associated renal disorders is not well known. The hAT1R gene has single nucleotide polymorphisms in its promoter that forms 2 haplotypes (Hap), Hap-I and Hap-II. Hap-I of AT1R gene is associated with hypertension in Caucasians. We have hypothesized here that age will alter the transcriptional environment of the cell and will regulate the expression of hAT1R gene in a haplotype-dependent manner. This could likely make subjects with Hap-I increasingly susceptible to age-associated, AT1R-mediated complications. METHOD We generated transgenic (TG) mice with Hap-I and Hap-II. Adults (10-12 weeks) and aged (20-24 months) TG male mice containing either Hap-I or Hap-II were divided into 4 groups to study (i) the age-associated and haplotype-specific transcriptional regulation of hAT1R gene and (ii) their physiological relevance. RESULTS In aged animals, TG mice with Hap-I show increased expression of hAT1R and higher blood pressure (BP); suppression of antioxidant defenses (hemoxygenase, superoxide dismutase) and antiaging molecules (ATRAP, Klotho, Sirt3); increased expression of pro-inflammatory markers (IL-6, TNFα, CRP, NOX1); and increased insulin resistance. In vivo ChIP assay shows stronger binding of transcription factor USF2 to the chromatin of Hap-I mice. CONCLUSION Our results suggest that in aged animals, as compared with Hap-II, the TG mice with Hap-I overexpress hAT1R gene due to the stronger transcriptional activity, thus resulting in an increase in their BP and associated renal disorders.

The transcriptional regulation of the human angiotensinogen gene after high-fat diet is haplotype-dependent: Novel insights into the gene-regulatory networks and implications for human hypertension.

Single nucleotide polymorphisms (SNPs) in the human angiotensinogen (hAGT) gene may modulate its transcription and affect the regulation of blood pressure via activation of the renin-angiotensin aldosterone system (RAAS). In this regard, we have identified polymorphisms in the 2.5 Kb promoter of the hAGT gene that form two haplotype (Hap) blocks: -6A/G (-1670A/G, -1562C/T, -1561T/C) and -217A/G (-532T/C, -793A/G, -1074T/C & -1178G/A). hAGT gene with Hap -6A/-217A (Hap I) is associated with increased blood pressure whereas, Hap -6G/-217G (Hap II) is associated with normal blood pressure in human subjects. Since RAAS over activity contributes to hypertension in obesity, we have made transgenic mice (TG) containing either Hap I or Hap II of the hAGT gene to understand the role of obesity on its transcriptional regulation. Although, a high-fat diet (60% Kcal from fat, 12 weeks) elevates hAGT and mAGT regardless of haplotype, this effect is significantly (p<0.05) accentuated in Hap I mice, in both adipose and liver tissues. Chromatin Immuno- precipitation (ChIP) assay shows an increased binding of transcription factors including, GR, CEBPβ and STAT3 to the chromatin of the Hap I TG mice after high-fat diet as compared to Hap II TG mice (p<0.05). Differential plasma levels of hAGT in Hap II and I mice, after high-fat diet, further corroborate the variable transcriptional regulation of the hAGT, governed by gene-haplotypes. Taken together, our results show that SNPs in the Hap-I of the hAGT gene promote high-fat diet-induced binding of transcription factors GR, CEBP-β and STAT3, which lead to elevated expression of the hAGT gene in hepatic and adipose tissues.