The Bradykinin B2 Receptor Gene Is a Target of Angiotensin II Type 1 Receptor Signaling Bing Shen,* † Lisa M. Harrison-Bernard, ‡ Andrew J. Fuller, † Vanessa Vanderpool,* Zubaida Saifudeen,* and Samir S. El-Dahr* *Department of Pediatrics, Section of Pediatric Nephrology, † Department of Physiology, Tulane University Health Sciences Center, and ‡ Department of Physiology, Louisiana State University Health Sciences Center, New Orleans, Louisiana Cross-talk between G protein– coupled receptors (GPCR) is known to occur at multiple levels, including receptor heterodimer- ization and intracellular signaling. This study tested the hypothesis that GPCR cross-talk occurs at the transcriptional level. It was demonstrated that the bradykinin B2 receptor gene (BdkrB2) is a direct transcriptional target of the angiotensin II (AngII) type 1 receptor (AT 1 R) in collecting duct cells. AngII induced BdkrB2 mRNA expression in mouse inner medullary collecting duct cells, and this effect was abrogated by AT 1 R blockade; in contrast, AT 2 R blockade was ineffective. Actinomycin D, an inhibitor of gene transcription, abrogated AngII-stimulated BdkrB2 expression. In addition, AngII produced dosage- and time-dependent increases in B2 receptor protein levels (2.9  0.4 fold; P < 0.05). AngII stimulated phosphorylation of cAMP response element binding protein (CREB) on Ser-133 and assembly of p-CREB on the BdkrB2 promoter in vivo. Moreover, AngII induced hyperacetylation of BdkrB2 promoter–associated H4 histones, a chromatin modification that is associated with gene activation. Mutations of the CRE abrogated AngII-induced activation of the BdkrB2 promoter. AngII-treated inner medullary collecting duct cells exhibited augmented intracellular calcium signaling in response to bradykinin, confirming the functional relevance of AT 1 -B2 receptor signaling. Finally, studies that were conducted in angiotensin type 1 receptor (Agtr1)-null mice revealed that BdkrB2 mRNA levels were significantly lower in the renal medulla of Agtr1 A / and Agtr1 A/B / than in Agtr1 / and Agtr1 B / mice. It is concluded that BdkrB2 is a downstream target of the AT 1 R-CREB signaling pathway. Transcriptional regulation represents a novel form of cross-talk between GPCR that link the renin- angiotensin and kallikrein-kinin systems. J Am Soc Nephrol 18: 1140 –1149, 2007. doi: 10.1681/ASN.2006101127 T he kallikrein-kinin and renin-angiotensin systems (KKS and RAS, respectively) are key regulators of cardiovas- cular and renal homeostasis. The balance between these two systems affects salt sensitivity, blood volume, vascular reactivity, and growth and development (reviewed in refer- ences [1–3]). The most widely recognized cross-talk between the KKS and RAS is at the level of the angiotensin-converting enzyme. More recently, physical and functional interactions between the receptors for angiotensin II (AngII) (AT 1 R) and bradykinin (B2R) have been reported, adding another layer of cross-talk between the RAS-KKS cascades. AT 1 R and B2R form heterodimers in cultured cells and intact tissues, and the recep- tor complex signals as a “super” AT 1A receptor (4,5). Infusion of subpressor dosages of AngII upregulates cardiac myocyte BdkrB2 gene expression in mice (6). Furthermore, treatment of vascular smooth muscle cells in culture with AngII produces a time-dependent induction of BdkrB2 mRNA, an effect that is inhibited by AT 1 R blockade (7). However, the mechanisms whereby AngII regulates BdkrB2 gene expression are not fully understood, and whether AT 1 R–B2R cross-talk operates in other AngII target tissues, such as the kidney, is not entirely clear. We previously demonstrated that the BdkrB2 promoter is regulated by a highly conserved cis-acting module that repre- sents the binding sites for the transcription factors p53 and cAMP response element binding protein (CREB), located at nucleotide positions -44 to -69 relative to the transcription start site (8 –10). The BdkrB2 modular enhancer drives reporter expression in mouse renal inner medullary collecting duct (IMCD3) cells but is considerably weaker in other cell types (10). Upon activation, CREB is phosphorylated on Serine 133 (p-CREB), a modification that facilitates recruitment of a co- activator, CREB-binding protein (CBP/p300) (11,12). In addi- tion to bridging CREB with the basal transcription machinery, CBP/p300 acetylates promoter-associated histones (13), relaxes local chromatin structure, and allows better access of transcrip- tion factors to the cis-regulatory elements (14). It has also been shown that p-CREB interacts with and recruits p53 to target promoters that contain composite target sites (15). Given that AngII stimulates the phosphorylation of p53 (on serine 15) (16,17) and CREB (on serine 133) (18), we hypothesized that Received October 18, 2006. Accepted January 25, 2007. Published online ahead of print. Publication date available at www.jasn.org. Address correspondence to: Dr. Samir S. El-Dahr, Department of Pediatrics, SL-37, Tulane University Health Sciences Center, 1430 Tulane Avenue, New Orleans, LA 70112. Phone: 504-988-377; Fax: 504-988-1852; E-mail: seldahr@tulane.edu Copyright © 2007 by the American Society of Nephrology ISSN: 1046-6673/1804-1140