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Circulation Research. 2006;99:1355-1366
Published online before print November 2, 2006, doi: 10.1161/01.RES.0000251700.00994.0d
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(Circulation Research. 2006;99:1355.)
© 2006 American Heart Association, Inc.

Molecular Medicine

A Novel Signal Transduction Cascade Involving Direct Physical Interaction of the Renin/Prorenin Receptor With the Transcription Factor Promyelocytic Zinc Finger Protein

Jan H. Schefe, Mario Menk, Jana Reinemund, Karin Effertz, Robin M. Hobbs, Pier Paolo Pandolfi, Patricia Ruiz, Thomas Unger*, Heiko Funke-Kaiser*

From the Center for Cardiovascular Research/Institute of Pharmacology (J.H.S., M.M., J.R., T.U., H.F.-K.), Department of Anesthesiology and Intensive Care Medicine (M.M.), and Center for Cardiovascular Research (K.E., P.R.), Charité–Universitätsmedizin Berlin, Germany; Max Planck Institute for Molecular Genetics (K.E., P.R.), Berlin, Germany; and Cancer Biology and Genetics Program (R.M.H., P.P.P.), Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York.

Correspondence to Heiko Funke-Kaiser, Center for Cardiovascular Research (CCR)/Institute of Pharmacology, Charité–Universitätsmedizin Berlin, Hessische Strasse 3-4, 10115 Berlin, Germany. E-mail heiko.funke-kaiser{at}charite.de


*    Abstract
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 *Abstract
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 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
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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{alpha} subunit of the phosphatidylinositol-3 kinase (PI3K-p85{alpha}). 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{alpha}. 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 • PLZF • ChIP • signal transduction


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Renin 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 function2,3 (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 to induce extracellular signal-regulated 1/2 phosphorylation.4

The mRNA of the receptor is highly expressed in brain, heart, and placenta with highest levels in the brain. In contrast, kidney, liver and pancreas show low mRNA expression levels.2,3 Via immunofluorescence, the receptor has been detected in mesangial and vascular smooth muscle cells of human heart and kidney.2,3 In addition, mRNA and protein expression have been demonstrated in macrophages, T cells, and granulocytes.4 The receptor is expressed on the cell surface in transfected mesangial cells,3 but there are also indications of an (additional) intracellular receptor localization.2,4

The cloned RER probably corresponds to the previously identified renin binding site on mesangial cells implicated in regulation of the plasminogen activator inhibitor-1 and hypertrophic effects.3,5 Very recently, it was shown that transgenic overexpression of the RER in smooth muscle cells causes a blood pressure elevation and an increase in heart rate.6

Concerning proteins encoded by the RER gene, it is of interest that the C-terminal part of the RER is identical to the vacuolar proton-translocating ATPase (V-ATPase) membrane sector–associated protein M8-9 (APT6M8-9, also known as ATP6AP2).7,8 V-ATPases, in general, exert several cellular functions such as neurotransmitter uptake and storage, endocytosis, and receptor recycling.9 Furthermore, RER and CAPER (homo sapiens endoplasmic reticulum [ER]-localized type I transmembrane adaptor precursor) are identical transcripts (GenBank accession no. AY038990). CAPER was identified in a yeast 2-hybrid screen using the ubiquitous tyrosine phosphatase PRL-1 (Phosphatase of Regenerating Liver-1) as bait (personal communication, R. Herbst, Palo Alto, LA). PRL-1 is involved in the regulation of cellular proliferation and transformation and exhibits a cell cycle–dependent subcellular localization, being localized to the ER in resting cells and to centrosomes and the spindle apparatus in mitotic cells.10

No direct protein interaction partner of the RER has been described so far. Therefore, the aim of this study was to analyze the signal transduction cascade of the RER based on protein-protein and downstream protein-DNA interactions, as well as on its subcellular localization. Furthermore, we studied the promoter architecture of the RER to mechanistically explain its cellular expression pattern.


*    Materials and Methods
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 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Yeast Two-Hybrid Screening
The complete coding sequence of the human RER, based on GenBank accession no. GI:21325928, was cloned into the yeast 2-hybrid bait-vector pBTM117c (kind gift from Erich Wanker, MDC, Berlin, Germany) expressing a LexA DNA-binding domain using the sense primer 5'-CATGGCTGTGTTTGTCGTGCTCCT-3' and the antisense primer 5'-TCAATCCATTCGAATCTTCTGG-3'. This construct was cotransformed into L40 yeast with a GAL4 activation domain fusion library of adult human heart cDNA in pACT2 vector (Clontech, Heidelberg, Germany). Yeast 2-hybrid screening was performed according to the cDNA library guidelines of the manufacturer. Positive clones were identified and checked after retransformation on minimal medium lacking tryptophan, leucine, and histidine and an additional ß-galactosidase assay.

All other methods are described in the online data supplement, available at http://circres.ahajournals.org.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
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mRNA Expression and Basal Promoter Analysis of Human RER
To analyze the expression pattern of the RER and to find appropriate cell lines for studying RER function, we performed a RT-PCR expression analysis of several different human neuronal, glial, epithelial, and endothelial cell lines as well as human kidney and human heart (as positive controls for expression of RAS components). This analysis showed a ubiquitous mRNA expression pattern of the RER in contrast to other components of the RAS (Figure 1A). In addition, we performed a Northern blot to assess the level of RER mRNA expression. RER mRNA can be easily detected by this method indicating strong expression of this gene (Figure 1B).


Figure 1
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  		Figure 1. A, RT-PCR expression analysis of the human renin/prorenin receptor and other components of the RAS. The indicated cell lines and tissues of human origin were analyzed by RT-PCR. Hypoxanthine guanine phosphoribosyltransferase (HPRT), GAPDH, and ß-actin served as positive controls. A reaction without addition of reverse transcriptase and a nontemplate control served as negative controls (data not shown). AT1R indicates AT1 receptor; AT2R, AT2 receptor; AGT, angiotensinogen; ACE, angiotensin-converting enzyme. B, Northern blot analysis of the human renin/prorenin receptor. Total RNA extracted from the indicated human cells and from human kidney was hybridized with a probe specific for human RER. Standardization was performed against human ß-actin.

To analyze the gene regulatory mechanisms responsible for the observed ubiquitous expression of the RER, we determined its transcriptional start sites and examined its promoter function. A RNA ligase-mediated 5' rapid amplification of cDNA ends (RACE) experiment, which only amplified capped mRNA, indicated multiple transcriptional start sites at positions 76, 86, 96, 102, 105, 112, and 125 bp upstream of the translational start codon (Figure 2A), which is consistent with the TATA box–less architecture of this promoter.11 In addition, 3 serial-deletion mutants of the human RER promoter (spanning 126, 500, and 1100 bp upstream of the translational start codon) were subcloned for luciferase reporter gene assay. The RER promoter is characterized by a very high promoter activity in the examined neuronal, endothelial, and epithelial cell lines (Figure 2B). Its activity is even higher than the activity of the human ECE-1c promoter, which was shown to be a strong, ubiquitous housekeeping promoter (Figure 2B).12,13 Additionally, our results indicate that 500 bp of the human RER promoter are sufficient to drive maximal promoter activity in these cell types (Figure 2B).


Figure 2
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  		Figure 2. A, Transcriptional start sites of the human renin/prorenin receptor. A RNA ligase-mediated 5' RACE was performed on SH-SY5Y cells, and the resulting PCR products were separated in an agarose gel (left) and subcloned for sequencing (right). No. 1 indicates 100 bp DNA ladder; 2, RACE reaction; 3, negative control. Transcriptional start sites are boxed and underlined; the translational start site "atg" is marked bold. The sequence shown corresponds to GenBank accession no. GI:37546587. B, Characterization of the human RER promoter in different human cells. Serial-deletion mutants of the human RER promotor were subcloned into the pGL3-basic luciferase reporter vector. Numbers indicate base pairs upstream from the translational start site. Standardization was achieved by cotransfection with phRL-null vector, which encodes renilla luciferase. Relative luciferase activity (RLA) indicates promoter activity relative to an insertless pGL3-basic vector. The strong housekeeping promoter of the human ECE-1c isoform (968 bp upstream of the translational start site) served as control (C).

Subcellular Localization of the RER
Because the subcellular localization of the RER may be a clue for the understanding of its function, we addressed this question using different methods. Initially, the localization of the RER within cellular membranes (ie, plasma membrane and/or membranes of organelles) was demonstrated by fractionated protein isolation followed by Western blotting of HeLa-S3 cells (Figure 3A). The observed molecular mass of approximately 38 to 39 kDa in the membrane fraction is consistent with the size of full-length RER described by Nguyen et al,3 whereas the lower band of approximately 35 to 36 kDa seen in cytosolic and membrane fractions (Figure 3A) likely corresponds to the {Delta}4-splice variant reported by Ramser et al,8 in which the 96 bp-sized exon 4 is missing.


Figure 3
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  		Figure 3. A, Western blot analysis of the human renin/prorenin receptor. Cytosolic (C) (12 µg) and membrane (M) (9 µg) proteins of HeLa-S3 cells were separated by SDS-PAGE, and Western blotting was performed using an antibody against human RER. B, Immunocytology of full-length RER. HeLa-S3 cells were transiently transfected with c-myc- and FLAG-tagged full-length RER, and subcellular localization was analyzed by immunofluorescence microscopy. Transfection of an insertless vector served as negative control. 4',6-Diamidino-2-phenylindole (DAPI) was used as nuclear marker. C, Fluorescence microscopy of EGFP-RER fusion proteins. A full-length wild-type RER (RER full), a full-length RER, in which the atypical ER-retention motif had been mutated (RER K/R mut), and the V-ATPase segment of the RER (RER ATPase), each C-terminally fused to EGFP, were transiently transfected into HeLa-S3 cells. Markers for the nuclear (DAPI), ER, and lysosomal compartment were used as indicated.

To further analyze the cellular membrane compartment in which the RER is localized, we performed a set of fluorescence microscopic experiments. Immunocytology of c-myc- and FLAG-tagged RER in HeLa-S3 cells indicated a perinuclear localization (Figure 3B). We next generated 3 different enhanced green fluorescence protein (EGFP) fusion proteins of the RER (each as N- and C-terminal fusion): (1) a full-length wild-type RER (RER full); (2) a full-length RER in which its atypical ER-retention motif KXXXX14,15 was mutated to RXXXX (RER K/R mut); and (3) the V-ATPase segment of the RER (RER ATPase). The full-length RER construct showed again a perinuclear localization and colocalized with a marker of the ER (Figure 3C). Mutagenesis of the ER retention motif resulted in a loss of perinuclear/ER localization (Figure 3C). Interestingly, the V-ATPase segment of the RER showed a different localization pattern compared with the full-length RER, as it was localized primarily to the lysosomal compartment (Figure 3C). EGFP N-terminally fused to these constructs yielded similar results (data not shown).

Protein-Protein Interaction Partners of the RER
No direct molecular interactions of the RER have been described so far. Therefore, a major objective of our study was to identify protein interaction partners of this ubiquitously expressed receptor to gain insight into its signal transduction cascade. For this purpose, we performed a yeast 2-hybrid screening using a human adult heart cDNA library (prey) and the full-length human RER (bait). The C-terminal third of the transcription factor PLZF (PromyeLocytic Zinc Finger protein) was identified as RER-interacting protein in 4 clones, 3 of which were independent. To confirm this RER-PLZF interaction, we performed a coimmunoprecipitation (CoIP) using transient transfections of full-length human RER and full-length human PLZF. Figure 4A demonstrates the ability of the RER to interact with PLZF in a system using tagged proteins. This finding was further confirmed in an endogenous context (Figure 4B).


Figure 4
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  		Figure 4. A, Confirmation of the RER-PLZF interaction by CoIP. HEK293 cells were transiently transfected with FLAG-tagged RER and/or c-myc–tagged PLZF. Immunoprecipitation and Western blotting were performed using anti-FLAG and anti–c-myc antibodies. Several controls were used as indicated. B, Endogenous interaction between RER and PLZF. Total lysate of HEK293 cells was subjected to immunoprecipitation using an anti-PLZF antibody or protein A agarose as control. Subsequent Western blotting of total lysate and immunoprecipitation eluates were performed as indicated. C, The C-terminal domain of the RER interacts with PLZF. HEK293 cells were cotransfected with pCEP4-PLZF-myc and deletion mutants of the RER fused to EGFP. RER full indicates full-length coding sequence; RER n-term del, deletion of the first N-terminal 16 aa; RER c-term del, deletion of the C-terminal (cytoplasmic) 18 aa; EGFP alone. CoIP was performed with an agarose-coupled anti–myc-antibody. Total lysates and immunoprecipitation eluates were subjected to Western blotting using anti–c-myc and anti-EGFP antibodies. D, The RER is able to form homodimers. The human RER was tagged with 2 different tags and transiently transfected into HEK293 cells. The ability of the RER to form homodimers was shown by CoIP.

In an additional CoIP experiment using C- and N-terminal deletion mutants of the RER, we identified the C-terminal, cytoplasmic region of the RER as the interaction domain for PLZF (Figure 4C).

Because dimerization is a common feature of several receptors, we investigated whether homodimerization may also be a characteristic of the RER. For this purpose, human RER constructs with 2 different tags were transiently transfected. CoIP experiments indicated that the RER is able to form homodimers (Figure 4D).

Functional Analysis of the Renin-RER-PLZF Signal Transduction Pathway
In initial experiments involving PLZF overexpression in HEK293 cells (which endogenously express RER and PLZF mRNA [data not shown]), we observed that RER mRNA is reduced by renin stimulation (to 80.6±3.3%) and PLZF transfection (to 72.6±5.2%), respectively (Figure 5A). A combination of both repressed RER mRNA to 45.4±4.7% (Figure 5A). We could substantiate these findings by using serial-deletion mutants of the human RER promoter as described above. Only a promoter construct comprising the region [–501; –1100] can be repressed by PLZF cotransfection (Figure 5B).


Figure 5
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  		Figure 5. A, Decrease of RER mRNA by renin stimulation and PLZF cotransfection. HEK293 cells with (pCEP4-PLZF) (+) or without (pCEP4 insertless) (–) PLZF overexpression were stimulated with renin (gray columns) or vehicle (white columns), and RER mRNA was quantified by real-time PCR. mRNA expression level of the first column was set to 100%. B, PLZF is able to repress RER promoter activity. SH-SY5Y and HEK293 cells were transfected with serial-deletion mutants of human RER promoter (numbers indicate promoter region relative to the translation start site in base pairs). Cotransfection was performed with a PLZF expression vector (gray columns) or an insertless control vector (pCEP4) (white columns). RER promoter activity was determined using a luciferase reporter assay. RLA indicates relative luciferase activity. C, Repression of RER promoter activity by renin stimulation requires RER and a PLZF cis element. HEK293 cells overexpressing PLZF (by transient transfection of a pCEP4-PLZF expression vector) were stimulated with renin (gray columns) or vehicle (white columns), and relative luciferase activity (RLA) of a wild-type RER promoter reporter construct (WT) or a RER promoter reporter construct with a mutation of the PLZF consensus sequence at position [–1097; –1083] (mut) was determined. –1110 indicates the length of the subcloned promoter relative to the ATG. The functional importance of the RER was examined by using siRNA against RER (+) compared with a control siRNA (–). D, Activation of PI3K-p85{alpha} promoter activity by renin stimulation requires RER. The activity of the PI3K-p85{alpha} promoter was analyzed in PLZF-overexpressing HEK293 cells in an experimental setting analogous to Figure 5C, using siRNA against the RER or control siRNA. E, Increase of PI3K-p85{alpha} mRNA by renin stimulation requires RER. HEK293 cells with (pCEP4-PLZF) or without (pCEP4 insertless) PLZF overexpression were stimulated with renin (gray columns) or vehicle (white columns), and PI3K-p85{alpha} mRNA (normalized to 18S rRNA) was determined by quantitative real-time PCR. The functional importance of the RER was analyzed by siRNA against RER (+) compared with a control siRNA (–). mRNA expression level of the first column was set to 100%.

Bioinformatic analysis of the RER promoter using MatInspector (http://www.genomatix.de)16 indicated the presence of a PLZF consensus sequence (5'-AACTACAGTTTTCAC-3') with a high core and matrix similarity located in the region [–1097; –1083] relative to the translational start codon of the promoter.

To investigate functional downstream effects of the RER-PLZF interaction and to analyze whether these are influenced by stimulation of the RER with its ligand, renin, we performed a set of experiments in which the different components of the putative renin-RER-PLZF pathway and the promoter of the human RER were experimentally modulated in HEK293 cells.

RER stimulation was performed by renin incubation; dependence on RER was analyzed by small interfering RNA (siRNA) against this receptor; the influence of PLZF was examined by cotransfection experiments with a PLZF expression vector (overexpression was confirmed by real-time PCR [data not shown]); and the functional importance of the PLZF consensus sequence within the human RER promoter was evaluated by site-directed mutagenesis of luciferase reporter constructs. siRNA against RER repressed RER mRNA to 29.8±5.2%, as shown by real-time PCR; a similar reduction was observed by Western blotting (data not shown). Readouts comprised RER promoter activity and mRNA level. The known PLZF target gene, the p85{alpha} subunit of the phosphatidylinositol-3 kinase (PI3K-p85{alpha}), served as an additional downstream candidate gene of a RER/PLZF pathway activation.17

Stimulation of the RER with renin resulted in a decrease in the activity of a wild-type RER promoter by approximately 30% compared with all controls (Figure 5C). Importantly, the presence of the RER was necessary for this effect (Figure 5C).

Consistent with this repressive effect of PLZF on the RER promoter, site-directed mutagenesis of the PLZF consensus sequence [–1097; –1083] caused a derepression (to >150%) of promoter activity. The effect of renin stimulation was also abolished by this mutagenesis (Figure 5C).

Similar but inverse effects were observed with respect to the PI3K-p85{alpha} promoter, which is known to be positively regulated by PLZF17. RER stimulation with renin caused an increase of 45% in PI3K-p85{alpha} promoter activity compared with control; this effect was abolished by downregulation of the RER using siRNA (Figure 5D).

To verify these findings, we analyzed the effect of RER stimulation on PI3K-p85{alpha} mRNA by real-time PCR analysis. Consistent with our promoter data, stimulation with renin increased PI3K-p85{alpha} mRNA in systems with and without PLZF overexpression by 105.2±6.74% or 30.2±7.9% (relative to vehicle control), respectively (Figure 5E). Again, downregulation of RER by siRNA abolished this induction regardless the expression level of PLZF (Figure 5E).

Translocation and Promoter Recruitment of PLZF on Renin Stimulation
Additionally, we examined whether activation of the RER by renin is able to cause a translocation of PLZF from the cytoplasm to the nucleus. The subcellular localization of c-myc–tagged PLZF was evaluated by Western blotting after fractionated extraction of nuclear and cytosolic proteins. Incubation of HEK293 cells with renin caused a clear increase in nuclear PLZF, whereas cytoplasmic PLZF almost disappeared (Figure 6A). Importantly, this translocation of PLZF again required the presence of the RER as indicated by our siRNA experiments (Figure 6A). Similar results were obtained in untransfected HEK293 cells using Western blotting against endogenous PLZF (data not shown).


Figure 6
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  		Figure 6. A, RER activation causes a translocation of PLZF to the nucleus. HEK293 were transfected with c-myc-tagged PLZF. Twenty-four hours later, siRNA directed against the RER (+), or a siRNA control (–), were applied. Forty-eight hours after siRNA transfection, cells were incubated with 10 nmol/L renin for 1 hour after a 24-hour starving period, followed by fractionated isolation of cytosolic and nuclear proteins. These proteins were subjected to SDS-PAGE and Western blotting. A Western blot directed against TATA box–binding protein (TBP) served as control for subcellular fractionation and loading. B, Renin stimulation increases recruitment of PLZF to the RER promoter. HEK293 cells were stimulated for 2 hours with renin or vehicle followed by ChIP using an anti-PLZF antibody. Recruitment of PLZF to the RER promoter was analyzed by real-time PCR and standardized to the recruitment of TBP to the ß-actin promoter. In addition, several controls were performed as described in detail in the Materials and Methods. C, PLZF protein binds to a consensus sequence within the human RER promoter. Nuclear proteins were extracted from native HEK293 cells. Electrophoretic mobility-shift assay was performed using oligonucleotides corresponding to the wild-type and a mutated form of the PLZF consensus sequence at position [–1097; –1083] in the human RER promoter. An oligonucleotide derived from the PI3K-p85{alpha} promoter served as control. S indicates specific band shift; SS1 and SS2, super-shifted band shift; ns, nonspecific band shift.

To evaluate the recruitment of endogenous PLZF to the human RER promoter within the chromatin context, we performed a chromatin-immunoprecipitation (ChIP) experiment. To ensure valid quantification of transcription factor recruitment, immunoprecipitated DNA was quantified applying real-time PCR analysis and multiple positive and negative controls. Renin stimulation of HEK293 cells did increase the PLZF recruitment to the RER promoter region encompassing the PLZF cis-element at position [–1097; –1083] approximately 6-fold (Figure 6B).

The binding of PLZF to this cis element of the human RER promoter (and not to a mutated form) was further confirmed by electrophoretic mobility-shift assay (Figure 6C), because we found a super-shifted band using a PLZF antibody. Furthermore, the observed pattern was identical compared with the positive control oligonucleotide derived from the PI3K-p85{alpha} promoter.17

Cellular Effects of the Renin-RER-PLZF Signal Transduction Pathway
To analyze the cellular effects of renin stimulation and to confirm the importance of PLZF and PI3Ks within the signal transduction cascade of the RER, we investigated the effects of renin, siRNA against PLZF, and pharmacological inhibition of PI3Ks by wortmannin in rat H9c2 cardiomyoblasts. Catalytic effects of renin incubation were excluded by blockade of the angiotensin AT receptor with 10 µmol/L losartan; mRNA of the AT2 receptor was not detectable in these cells using real-time PCR analysis (data not shown).

Renin stimulation increased the number of viable cells to 133.9% compared with vehicle control. Preincubation with wortmannin partly abolished this effect, whereas siRNA against PLZF completely abrogated proproliferative effects of renin stimulation (Figure 7A). Consistent results were observed regarding apoptosis using caspase 3 and 7 activity as readout. Renin stimulation decreased caspase activity to 45.9% (in comparison to vehicle). Wortmannin partly and PLZF siRNA completely abolished this effect (Figure 7B).


Figure 7
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  		Figure 7. A, The increase in cell number induced by renin is mediated by PLZF and, partly, PI3Ks. Rat H9c2 cardiomyoblasts were transfected with siRNA against PLZF (+) or scrambled control siRNA (–). Forty-eight hours later, after 24 hours of serum starving, cells were pretreated with 10 µmol/L losartan (AT1 receptor antagonist), and, additionally, 1 µmol/L wortmannin (PI3K inhibitor) (+) or vehicle (–). Thirty minutes later, cells were stimulated with 10 nmol/L renin (+) or vehicle (–). The number of viable cells was determined using an ATP-dependent luciferase reaction. B, The decrease in apoptosis induced by renin is mediated PLZF and, partly, PI3Ks. Experimental conditions were identical to A. The activity of caspase 3 and 7 was determined using a luciferase reaction and served as a measure of apoptosis. C, The decrease in RER mRNA expression induced by renin is mediated by PLZF. Experimental conditions were identical to A. RER mRNA (normalized to 18S rRNA) was determined by quantitative real-time PCR. D, The increase in PI3K-p85{alpha} mRNA expression induced by renin is mediated by PLZF. Experimental conditions were identical to A. PI3K-p85{alpha} mRNA (normalized to 18S rRNA) was determined by quantitative real-time PCR.

To confirm the gene regulatory downstream effects of RER activation in cardiomyoblasts, we also measured RER and PI3K-p85{alpha} mRNA in this experimental setting. RER mRNA decreased to 53.7% under renin stimulation compared with vehicle control. siRNA against PLZF (which repressed PLZF mRNA to 15.23±9.8% as shown by real-time PCR) reconstituted RER mRNA to 86.5% (Figure 7C). Renin stimulation increased PI3K-p85{alpha} mRNA to 224.2% compared with vehicle control. siRNA against PLZF abolished this effect (Figure 7D). Wortmannin did not affect RER or PI3K-p85{alpha} mRNA levels, respectively (Figure 7C and 7D).

RER and PI3K-p85{alpha} mRNA Expression in PLZF Knockout Mice
To substantiate our findings concerning the transcriptional regulation of RER and PI3K-p85{alpha} by PLZF in vivo, we quantified the respective transcript levels in PLZF knockout (PLZF–/–) and wild-type (WT) control mice, respectively. PLZF was not detectable in kidney and liver of WT and PLZF–/– mice. Within these organs, we did not observe any difference in mRNA levels of RER and PI3K-p85{alpha}. In contrast, a significant upregulation of RER mRNA (to 221.9% and 229.5% in heart and brain, respectively) and a significant downregulation of PI3K-p85{alpha} mRNA (to 71.8% and 48.6% in heart and brain, respectively) were detected in heart and brain of PLZF–/– mice compared with WT mice, which endogenously express PLZF in these organs (Figure 8A).


Figure 8
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  		Figure 8. A, PLZF regulates RER and PI3K-p85{alpha} mRNA in vivo. RNA of indicated tissues was extracted from homozygous PLZF knockout mice (PLZF–/–) and wild-type controls. mRNA levels of PLZF, RER, and PI3K-p85{alpha} (normalized to 18S rRNA) were measured by quantitative real-time PCR. Data represent mean value of 3 animals in each group±SD. Expression levels of wild-type mice were set to 100% in each organ. B, Signal transduction of the RER.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
In this work, we identified the transcription factor PLZF as the first direct protein-protein interaction partner of the human renin/prorenin receptor (RER). Furthermore, we describe a novel, short-loop signal transduction pathway involving renin, RER, the transcription factor PLZF, and 2 direct downstream targets, PI3K-p85{alpha} and the RER itself (Figure 8B). This pathway has been confirmed in human epithelial and rat cardiomyoblasts.

Stimulation of the RER with its ligand renin causes a translocation of PLZF from the cytoplasm to the nucleus and a recruitment of PLZF to promoter regions of PI3K-p85{alpha} and the RER. Depending on the promoter context, PLZF is able to activate the transcription of PI3K-p85{alpha} and to repress the gene expression of the RER. This dual role of PLZF as activator17,18 and also repressor19,20 has been described before but, to our knowledge, not in a single cellular context. The fascinating and uncommon observation that a receptor directly interacts with a transcription factor speaks for an extraordinary short signal transduction cascade. Similar mechanisms, by which a transcription factor or nuclear factor is directly activated at a receptor site or at an extranuclear membrane, have been described for the SMAD, JAK-STAT, Notch, and SREBP pathways.21,22 Remarkably, PLZF itself represses the promoter of its own direct interaction partner RER, thereby establishing a very short negative feedback loop. The observation that a RER promoter with a mutated PLZF cis element is refractory to effects of renin stimulation (Figure 5C) indicates that this DNA motif is necessary for this feedback mechanism. Binding of PLZF to this motif was further confirmed by our electrophoretic mobility-shift assay experiments (Figure 6C). Most importantly, the recruitment of native PLZF to the corresponding cis-element region of the RER promoter was demonstrated in the chromatin context by ChIP, with a significant increase under renin stimulation (Figure 6B). The ability of renin to decrease the gene expression of RER and to increase the gene expression of PI3K-p85{alpha} depends on the presence of the RER. Therefore, our data prove the existence of the RER as a functional renin receptor, thereby confirming the work of Nguyen et al.3

Furthermore, we were able to demonstrate cellular effects of this novel renin-RER-PLZF signal transduction pathway. RER activation caused a significant increase in cell number and a concomitant decrease in apoptosis of cardiomyoblasts. These effects are mainly mediated by PLZF as shown by siRNA experiments. PI3K inhibition did affect these cellular effect to a minor degree, consistent with PI3K-p85{alpha} being a downstream target of PLZF. These effects are in accordance with the known physiological role of PLZF as discussed below. Additionally, several studies have shown that PI3Ks contribute to cardiac hypertrophy involving increase in protein synthesis and activation of the Akt pathway.17,23,24 Finally, we could demonstrate that the RER-PLZF pathway also acts in vivo. Experiments in tissues derived from PLZF knockout mice clearly show that absence of PLZF causes a derepression of RER mRNA and a decrease in PI3K-p85{alpha} mRNA in organs endogenously expressing PLZF.

The primary perinuclear subcellular localization of the RER observed in our work conflicts with the plasma membrane localization described by Nguyen et al.3 Nevertheless, all of our experiments, including use of different constructs, mutagenesis, and colocalization studies, indicated an intracellular localization of the RER. Furthermore, the total cellular membrane lysates used for their kinetic studies may still contain intracellular membrane proteins and, therefore, do not exclude an intracellular localization of the RER. The possibility that our results concerning the perinuclear localization are caused by an artifact related to tags or overexpression is unlikely because we used several different C-terminal and also N-terminal tags. In addition, mutagenesis of the atypical C-terminal ER-retention signal strongly reduced the perinuclear localization of the RER. The fact that CAPER, which is identical to the RER as discussed above, can directly bind to PRL-1 (a protein observed in the ER in nonmitotic cells) also supports our results.

On the other hand, our experiments indicate that an extracellular signal (renin) can affect the signal transduction of the RER despite its mainly intracellular localization. Several mechanisms might account for this observation. Firstly, other renin-binding receptors, such as the mannose-6-phosphate receptor, could internalize renin and prorenin.25 Secondly, a nonsecreted (ie, intracellular) renin isoform, which could directly interact with an intracellular RER, has been described containing an alternative first exon termed (albeit identical) "exon 1b"26,27 and "exon 1A,"28,29 respectively. Intracellular renin administration was reported to increase inward calcium current in cardiomyocytes.30 In addition, transgenic overexpression of the nonsecreted, intracellular form of renin and angiotensinogen in the brain causes an increase in drinking volume and mean arterial pressure.31 Thirdly, it could be possible that very small amounts of the RER within the plasma membrane are sufficient for the initiation of a RER signal transduction cascade. In this context, the observed homodimerization of the RER might also be of importance, because dimerization can affect subcellular localization.32

In this study, we were able to demonstrate a ubiquitous expression of the human RER, a high promoter activity in different cell types and multiple transcriptional start sites in a TATA box–less promoter. These features are consistent with housekeeping properties of the RER gene,13 suggesting basal cellular functions of this protein. In this context, it is of interest that the C-terminal part of the RER is, as discussed above, identical to the V-ATPase membrane sector-associated protein M8-9, because V-ATPase have functions in almost every eukaryotic cell9. Nevertheless, APT6M8-9 only constitutes 69 to 100 aa (GenBank accession no. GI:5031590)7 of the 350 aa3 of the full-length renin receptor. Therefore, the functions of the RER exceed those of a pure V-ATPase subunit, which is also supported by the data on the signal transduction and the different subcellular localizations of full-length RER and APT6M8-9 provided here. The mechanism by which the APT6M8-9 protein is generated, remains to be clarified. Alternative promoters and posttranslational cleavage by an unidentified protease are putative explanations.

The transcription factor PLZF contains multiple zinc-finger domains and is disrupted in patients with acute promyelocytic leukemia (APL) caused by t(11;17)(q23;q21) chromosomal translocation.33,34 This APL subform is characterized by PLZF-RAR{alpha} (PLZF-retinoic acid receptor-{alpha}) fusion proteins, which recruit histone deacetylase 1 (HDAC1) and do not respond to retinoic acid any more, explaining the missing response of these patients to retinoic acid treatment.34–36

Wild-type PLZF can act as growth repressor and exerts proapoptotic functions during development.33,37 Concerning the RAS, it seems important to note that PLZF was recently described as an adaptor protein of the AT2 receptor in the heart.17 This direct PLZF-AT2 receptor interaction was associated with stimulation of protein synthesis and putative cardiac hypertrophy.17

Data obtained from PLZF knockout mice indicate that this transcription factor is involved in limb and axial skeletal patterning,37 whereas the brain phenotype of these mice was not described by the authors. Consistent with this observation, PLZF target genes include hox genes,37–39 besides the p85{alpha} subunit of PI3K17 and cyclin A2.20

Concerning the biological significance of the RER beyond its possible role in the RAS, it was recently shown by Ramser et al that a mutation in the renin receptor gene is a cause of X-linked mental retardation (XLMR) and epilepsy (XMRE) syndrome in humans.8 Consistent with this observation are the results of a zebrafish mutagenesis screen, in which a mutation in the ATP6AP2 (which is identical to RER) gene caused a reduction in head size and necrosis of the central nervous system.40 Remarkably, the human RER mutation observed by Ramser et al altered neither binding affinity for renin nor the RER-mediated augmentation of the catalytic efficiency of renin for angiotensinogen cleavage.8 In addition, the effect of this mutation (and also the effect of wild-type RER stimulation by renin3) on mitogen-activated protein kinase signaling are only modest,8 if non-significant. Therefore, it could be possible that the human RER mutation exerts its effect by an altered PLZF-mediated signal transduction. The complex, dynamic expression of PLZF in the developing central nervous system, and its likely role in hindbrain and also forebrain segmentation supports this notion.41,42

Related to the role of the RER in central nervous system development is the observation that RER mRNA can be detected in human glioblastomas as well as in glioblastoma cell lines and that renin inhibitors can reduce the cell number in glioblastoma cell lines. This is probably caused by modulation of RER function, because this effect is independent of AT1 and AT2 receptor activity.43

The clinical relevance of the RER is underlined by the interesting observation that a decoy deca-peptide corresponding to the handle region of prorenin, which competitively inhibits prorenin binding to its receptor, attenuated the development and progression of cardiac fibrosis44 and also inhibited the development of diabetic nephropathy in rat models.45 Remarkably, a recent study demonstrated the ability of RER activation to upregulate transforming growth factor-ß1 in mesangial cells independent of angiotensin II actions.46

Knowledge regarding the signal transduction of the RER is of importance to evaluate the efficacy and safety of renin inhibitors, such as aliskiren, which is currently in phase III clinical trials.47 As expected, renin inhibitors reduce plasma renin activity (ie, enzyme activity with respect to angiotensin I generation). Nevertheless, they increase total amount of plasma renin protein, the RER ligand, dramatically (up to 34-fold48). Therefore, it is crucial to examine whether renin inhibitors change the intrinsic activity of renin with respect to the RER. In this context, it is important to note that overactivation of the RER might be deleterious (eg, with respect to end-organ damage) considering the activation of mitogen-activated protein kinases and PLZF downstream of the RER as well as the effects of the handle region decoy peptide mentioned above. On the other hand, a blockade of the RER signal transduction might be harmful, at least in pregnant women, because of the developmental importance of the RER and its direct interaction partner PLZF.

To conclude, our results demonstrate the existence of a novel signal transduction pathway downstream of the human renin/prorenin receptor, which involves direct binding of the transcription factor PLZF to the receptor, its translocation to the nucleus, and the positive and negative regulation of target genes. Based on the already described biomedical relevance of the RER and PLZF, respectively, this pathway, connecting both molecules, might be of importance in human physiology and pathophysiology.


*    Acknowledgments
 
Sources of Funding

This project was supported by a Forschungsförderung of the Charité–Universitätsmedizin Berlin.

Disclosures

T.U. is member of the Aliskiren International Advisory Board of Novartis Pharma AG (Basel, Suisse).


*    Footnotes
 
*Both authors contributed equally to this work. Back

Original received July 17, 2006; revision received October 9, 2006; accepted October 25, 2006.


*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
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 *References
 

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