The Zinc Finger-Only Protein Zfp260 Is a Novel Cardiac Regulator and a Nuclear Effector of α1-Adrenergic Signaling

Abstract

␣1-Adrenergic receptors mediate several biological effects of catecholamines, including the regulation of myocyte growth and contractility and transcriptional regulation of the atrial natriuretic factor (ANF) gene whose promoter contains an ␣1-adrenergic response element. The nuclear pathways and effectors that link receptor activation to genetic changes remain poorly understood. Here, we describe the isolation by the yeast one-hybrid system of a cardiac cDNA encoding a novel nuclear zinc finger protein, Zfp260, belonging to the Krüppel family of transcriptional regulators. Zfp260 is highly expressed in the embryonic heart but is downregulated during postnatal development. Functional studies indicate that Zfp260 is a transcriptional activator of ANF and a cofactor for GATA-4, a key cardiac regulator. Knockdown of Zfp260 in cardiac cells decreases endogenous ANF gene expression and abrogates its response to ␣1-adrenergic stimulation. Interestingly, Zfp260 transcripts are induced by ␣1-adrenergic agonists and are elevated in genetic models of hypertension and cardiac hypertrophy. The data identify Zfp260 as a novel transcriptional regulator in normal and pathological heart development and a nuclear effector of ␣1-adrenergic signaling.The endogenous catecholamines epinephrine and norepinephrine are key regulators of numerous physiologic functions, including learning, memory, and cardiovascular and endocrine homeostasis. Their dysregulation has been implicated in human conditions such as depression and addiction and in cardiovascular and metabolic diseases. Their effects are mediated by three classes of adrenergic receptors (ARs), ␤, ␣1, and ␣2, each comprised of three distinct gene products. They all belong to the superfamily of seven transmembrane G-protein-coupled receptors. ␣1-ARs are critical for a variety of catecholamine actions such as the control of blood pressure, smooth muscle contraction, myocardial function, and glycogenolysis. The importance of ␣1-ARs in physiology and pathophysiology is evidenced by the wide clinical use of ␣1-AR agonists and antagonists for the treatment of cardiovascular disease, flu and allergy symptoms, and benign prostate hyperplasia (40, 43). Paradoxically, the molecular mechanisms underlying ␣1-AR action remain undefined.Historically, the role of ␣1-ARs in different biologic systems was largely inferred from pharmacologic studies, but the development of transgenic mice with targeted deletion or overexpression of specific ␣1-AR subtypes has further confirmed the essential role of specific ␣1-ARs in regulation of physiologic processes (reviewed in references 40 and 44). For example, ␣1-null mice rapidly develop hyperinsulinemia, insulin resistance, and obesity in response to high-fat feeding, confirming the important role of ␣1-AR in the regulation of glucose homeostasis (7). The use of genetically altered mice also confirmed the essential role of ␣1-ARs in mediating the effects of some psychostimulants and opiates and, more generally, their involvement in the regulation of vario...

Dissociation of Cardiogenic and Postnatal Myocardial Activities of GATA4

“…NIH 3T3 cells were cultured and transfected, and luciferase assays using ANF reporter plasmids and GATA4, NKX2.5, Tbx5, MEF2C, and SRF expression vectors were carried out as previously described (8,10). All experiments were repeated at least three times in duplicate.…”

“…Purified recombinant adenoviral plasmid DNA was digested with PacI to expose the terminal repeats and was later transfected to AD-293 cells in which deleted viral assembly genes are complemented in vivo. The viruses were produced in bulk, and titers were determined as reported previously (8).…”

“…Adenoviral constructs expressing HA-tagged GATA4 (GATA4 wild type [WT], GATA4 with the S105A mutation [GATA4-S105A], and GATA4 consisting of residues 201 to 440 [GATA4(201-440)]) in recombinant replication-deficient type 5 adenovirus (Ad5) were generated using an AdEasy XL adenoviral vector system (Stratagene). cDNAs of the corresponding rat GATA4 constructs were subcloned into Ad5 shuttle vector pshuttle using HindIII and EcoRV, and the adenoviruses were generated through recombination with the pAdEasy-1 as described previously (8). Transformant bacteria were selected for kanamycin resistance, and recombination was identified via restriction enzyme digestion.…”

“…Treatment with doxorubicin (Dox) was as described previously (1). Immunofluorescence on cardiomyocytes was performed as previously described (8), using a rabbit polyclonal HA antibody (dilution, 1/500) from Santa Cruz and phalloidin-Alexa Fluor 488 (dilution, 1/400). Apoptosis was detected by a terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay as recommended in the Apoptag kit (Chemicon).…”

Transcriptional Regulation of the Fetal Cardiac Gene Program

“…1) (11,12). These elements contribute singly and/or cooperatively to the basal and inducible activation of ANP promoter activity in cardiac cells (13).…”