Abstract-A human renin/prorenin receptor (RER) has recently been cloned. To gain insight into the molecular function of the RER, we studied its signal transduction mechanisms. Initially, we found a ubiquitous and intracellular expression pattern of the human RER. Consistently, we observed several transcriptional start sites and a high promoter activity of the human RER. We could identify the transcription factor promyelocytic zinc finger (PLZF) protein as a direct protein interaction partner of the C-terminal domain of the RER by yeast 2-hybrid screening and coimmunoprecipitation. Coimmunoprecipitation experiments also indicated homodimerization of the RER. On activation of the RER by renin, PLZF is translocated into the nucleus and represses transcription of the RER itself, thereby creating a very short negative feedback loop, but activates transcription of the p85␣ subunit of the phosphatidylinositol-3 kinase (PI3K-p85␣). Small interfering RNA against the RER abolished these effects. A PLZF cis-element in the RER promoter was identified by site-directed mutagenesis and electrophoretic mobility-shift assay. Renin stimulation caused a 6-fold recruitment of PLZF to this promoter region as shown by chromatin immunoprecipitation. Moreover, renin stimulation of rat H9c2 cardiomyoblasts induced an increase of cell number and a decrease of apoptosis. These effects were partly abolished by PI3K inhibition and completely abrogated by small interfering RNA against PLZF. Finally, experiments in PLZF knockout mice confirmed the role of PLZF as an upstream regulator of RER and PI3K-p85␣. Our data demonstrate the existence of a novel signal transduction pathway involving the ligand renin, RER, and the transcription factor PLZF, which is of physiological and putative pathophysiological relevance. Key Words: renin receptor Ⅲ PLZF Ⅲ ChIP Ⅲ signal transduction R enin and prorenin are classically thought of as (pro)enzymes of the renin/angiotensin system (RAS), but recent evidence suggests that they also act as hormones because of their ability to bind cellular targets. 1 In 2002 a human renin/prorenin receptor (RER) has been cloned, which consists of 350 amino acids with a single transmembrane domain and specifically binds prorenin and renin. Interestingly, this receptor exerts a dual molecular function 2,3 (1) Binding of renin to its receptor increases the catalytic activity of renin approximately 4-to 5-fold. Furthermore, prorenin, which does not exhibit significant ability to generate angiotensin I in solution, gains enzymatic activity comparable to renin by binding to the RER, ie, the receptor is able to unmask the catalytic activity of prorenin. (2) The RER is also able to induce a signal transduction cascade on ligand binding. Binding of renin and also prorenin causes phosphorylation of the receptor and activation of the mitogen-activated protein kinases ERK 1 and 2 (extracellular signal-regulated kinases 1 and 2), whereas intracellular calcium or cAMP levels are not altered. Remarkably, even deglycosylated renin is able t...

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Transposon-based gene vectors have become indispensable tools in vertebrate genetics for applications ranging from insertional mutagenesis and transgenesis in model species to gene therapy in humans. The transposon toolkit is expanding, but a careful, side-by-side characterization of the diverse transposon systems has been lacking. Here we compared the Sleeping Beauty (SB), piggyBac (PB), and Tol2 transposons with respect to overall activity, overproduction inhibition (OPI), target site selection, transgene copy number as well as long-term expression in human cells. SB was the most efficient system under conditions where the availability of the transposon DNA is limiting the transposition reaction including hard-to-transfect hematopoietic stem/progenitor cells (HSCs), and the most sensitive to OPI, underpinning the need for careful optimization of the transposon components. SB and PB were about equally active, and both more efficient than Tol2, under nonrestrictive conditions. All three systems provided long-term transgene expression in human cells with minimal signs of silencing. Indeed, mapping of Tol2 insertion sites revealed significant underrepresentation within chromosomal regions with H3K27me3 histone marks typically associated with transcriptionally repressed heterochromatin. SB, Tol2, and PB constitute complementary research tools for gene transfer in mammalian cells with important implications for fundamental and translational research.

Background-Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited disorder that causes sudden death and right ventricular heart failure in the young. Clinical data suggest that competitive sports may provoke ARVC in susceptible persons. Genetically, loss-of-function mutations in desmosomal proteins (plakophilin, desmoplakin, or plakoglobin) have been associated with ARVC. To test the hypothesis that reduced desmosomal protein expression causes ARVC, we studied the cardiac effects of heterozygous plakoglobin deficiency in mice. Methods and Results-Ten-month-old heterozygous plakoglobin-deficient mice (plakoglobin ϩ/Ϫ ) had increased right ventricular volume, reduced right ventricular function, and spontaneous ventricular ectopy (all PϽ0.05). Left ventricular size and function were not altered. Isolated, perfused plakoglobin ϩ/Ϫ hearts had spontaneous ventricular tachycardia of right ventricular origin and prolonged right ventricular conduction times compared with wild-type hearts. Endurance training accelerated the development of right ventricular dysfunction and arrhythmias in plakoglobin ϩ/Ϫ mice. Histology and electron microscopy did not identify right ventricular abnormalities in affected animals. Conclusions-Heterozygous plakoglobin deficiency provokes ARVC. Manifestation of the phenotype is accelerated by endurance training. This suggests a functional role for plakoglobin and training in the development of ARVC.