doi: 10.1161/hq0202.104062
© 2002 American Heart Association, Inc.
Vascular Biology |
Identification of a Zinc Finger Homoeodomain Enhancer Protein After AT2 Receptor Stimulation by Differential mRNA Display
From the Department of Physiology (M.S.), Medical College of Wisconsin, Milwaukee; Knoll AG (A.W.A.H., U.J.), Ludwigshafen, Germany; Department of Cardiology (B.S.), Medizinische Hochschule Hannover; and Institute of Pharmacology (M.S., Y.Z., J.W.F., T.U.), Christian-Albrechts-University Kiel, Kiel, Germany.
Address correspondence to Monika Stoll, PhD, Institute of Pharmacology, Christian-Albrechts-University Kiel, Hospitalstr 4, D-24105 Kiel, Germany. E-mail stoll{at}pharmakologie.uni-kiel.de
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Abstract |
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Using differential mRNA display, we isolated differentially expressed genes after stimulation of angiotensin II (Ang II) type 2 (AT2) receptors in PC12w cells. Among the polymerase chain reaction products isolated, we identified Zfhep, a zinc finger homoeodomain enhancer–binding protein and the angiotensin AT2 receptor itself, both implicated in developmental processes. In the presence of epidermal growth factor (EGF) a concordant expression pattern of Zfhep and AT2 mRNA was observed with marked downregulation 6 hours after stimulation with EGF+Ang II. In quiescent PC12w cells, Zfhep mRNA was time-dependently induced by Ang II (10-7 mol/L) up to 3 hours, an effect that was blocked by the selective AT2 antagonist PD123177 (10-6 mol/L). We extended our studies on Zfhep mRNA expression to cells of vascular origin (coronary endothelial cells [CECs]), that express AT1 and AT2 receptors, and that respond differently to Ang II dependent on which receptor subtype is stimulated. In CECs, Zfhep mRNA expression was induced up to 3 hours after AT2 stimulation. Interestingly, this Zfhep induction was observed only when the AT1 receptor was blocked by using the selective AT1 receptor antagonist, losartan (10-5 mol/L), suggesting a negative-regulatory influence of AT1 receptors on AT2-induced Zfhep mRNA induction. In conclusion, Zfhep not only represents a suitable marker for AT2 receptor activation, but it may also link AT2 signaling and downstream events involved in the proposed function of the AT2 receptor in development and regeneration.
Key Words: angiotensin II • Zfhep • differential mRNA display • angiotensin receptors
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Introduction |
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Angiotensin II (Ang II) exerts a remarkable diversity of physiological actions. In addition to its hemodynamic effects, Ang II has been implicated in the regulation of cell growth and differentiation.1–3 Two Ang II receptor subtypes have been defined, which are designated as AT1 and AT2 receptors.4 Most of the known hemodynamic and the growth-promoting effects of Ang II are mediated by AT1 receptors.5 Considerably less is known about the physiological role of AT2 receptors. Recent evidence suggests an involvement of AT2 receptors in development and apoptosis in various tissues6–9 as well as regeneration after injury.10–12 Although the signaling cascade of the AT1 receptor is well characterized, the signaling of the AT2 remains a matter of great debate. Overall, diverse putative AT2 receptor signaling pathways are discussed, which include activation of serine/threonine phosphatase PP2A and subsequent opening of the delayed rectifier K+-channel, activation of cytosolic phosphotyrosine phosphatases which may lead to closing of the T-type Ca2+ channel, inactivation of mitogen-activated protein kinase, presumably through PP2A and other phosphotyrosine phosphatases, and the activation of phospholipase A2 (for a review, see de Gasparo et al5). A hallmark of AT2 signaling appears to be the activation of phosphatases to inhibit phosphorylation steps in, eg, growth signaling.13,14 Recently, ceramide, which is also linked to phosphatase activation, was proposed to be the second messenger in AT2 receptor–mediated apoptosis.15,16 However, little is known concerning target genes of the AT2-signaling cascade. The AT2 receptor may exert its growth-modulating actions on various levels including the modulation of the signaling of other receptors17 and the activity of growth and transcription factors, as well as remodeling of the extracellular matrix. To search for downstream targets that mediate the growth-modulating properties of AT2 receptors, we used differential mRNA display18 to identify genes regulated after stimulation of AT2 receptors under conditions in which the known antiproliferative actions of this receptor subtype are well established.
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Methods |
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Cell Culture
PC12w cells, a substrain from a clonal isolation of a rat adrenal chromaffin cell tumor, were cultured in Dulbecco’s Modified Eagle Medium supplemented with 100U/100 µg/mL penicillin/streptomycin, 2 mmol/L L-glutamine (Gibco BRL), 5% heat inactivated fetal calf serum (Biowhitaker), 10% heat inactivated donor horse serum (Bioconcept) as previously described.19 Rat coronary endothelial cells (CECs) were isolated from male Wistar rats and characterized and cultured as previously described.3 To achieve quiescence, PC12w cells and CECs were serum-deprived for 48 hours before the experiment as previously described.3,19 PC12w cells were used from passages 8 to 15, and CECs were used exclusively at passage 2.
Angiotensin Receptor Binding Studies
The presence of angiotensin receptors was verified by binding studies as previously described.3,19
Experimental Protocol
For the initial differential mRNA display, quiescent PC12w cells were stimulated with epidermal growth factor (EGF, 50ng/mL) in the presence or absence of Ang II (10-7 mol/L). Total RNA was extracted 1 and 6 hours after stimulation of the cells while vehicle-treated cells served as controls.
To verify differential expression of the clones of interest, PC12w cells were stimulated in subsequent experiments with either EGF (50 ng/mL) or Ang II (10-7 mol/L) alone or in combination, and in the presence or absence of the specific AT1 receptor antagonist losartan (10-5 mol/L), and the selective AT2 receptor antagonist PD123177 (10-6 mol/L). Gene expression was investigated at time points 1, 3, and 6 hours after stimulation of cells.
In a second set of experiments, we investigated the gene expression of one of the clones isolated, Zfhep, in rat CECs. The experimental protocol for stimulation of CECs was identical to the protocol used for our studies on the growth-modulating actions of AT1 and AT2 receptors in these cells.3 In brief, CECs were serum-deprived for 48 hours and then stimulated with Ang II (10-7 mol/L) in the presence or absence of losartan (10-5 mol/L) and PD123177 (10-6 mol/L). In a second set of experiments, quiescent CECs were stimulated to proliferate for 16 to 20 hours by the addition of 25 ng/mL basic fibroblast growth factor (bFGF) before stimulation with Ang II in the presence or absence of the respective antagonists. Total RNA was extracted 1, 3, and 6 hours after stimulation of the cells with Ang II±antagonists.
Differential mRNA Display
Total RNA was extracted according to the protocol by Chomczynski and Sacchi20 and dissolved in water at a final concentration of 0.2 µg/µL. First-strand synthesis (5 µg total RNA) was performed with the Superscript Preamplification Kit (Gibco BRL) by using Oligo-dT12–18 oligonucleotides. Quality of reverse-transcribed cDNA (absence of contamination with genomic DNA and equality of cDNA amount) was verified by reverse-transcription–polymerase chain reaction (RT-PCR) by using intron spanning primers for ß-actin (Clontech). Second-strand cDNA synthesis was initiated by arbitrary priming with a modified primer based on the method described by Welsh et al,18 in which the original primer KZ (5'-CCCATGTGTACGCGTGTGGG-3') was modified by the insertion of a PstI restriction site, resulting in the primer AZ-PstI (5'-CCCTGCAGTGTACGCGTGTGGG-3'). In a final reaction volume of 100 µL, PCR was performed by using 200 µmol/L of each dNTP, 3 mmol/L of MgCl2, 400 nmol/L of each primer, and 2.5 U of Taq polymerase (Gibco BRL), corresponding PCR buffer, and 10 µCi of 
-32P-dCTP. The PCR temperature profile used was 94°C (5 minutes) hot start, low stringency annealing at 40°C (5 minutes), and primer extension at 72°C (5 minutes) followed by 10 cycles of 94°C (1 minute), 50°C (1 minute), 72°C (1 minute), and 30 cycles of 94°C (1 minute), 60°C (1 minute), 72°C (1 minute), and 5 minutes at 72°C to ensure double-stranded cDNA.
Gel Electrophoresis
Labeled products were displayed in duplicate on a 6% denaturing polyacrylamide sequencing gel, electrophoresed at 1500 V until the xylene cyanol dye had reached the bottom of the gel, and exposed to a x-ray film (Kodak) to visualize bands. Unique bands were excised, eluted from the gel, and re-amplified with the Az-PstI primer. PCR conditions for re-amplification were as follows: In a final reaction volume of 100 µL, PCR was performed by using 200 µmol/L of each dNTP, 2.5 mmol/L of MgCl2, 200 nmol/L of each primer, and 2.5 U of Taq polymerase plus corresponding PCR buffer. The PCR temperature profile used was 94°C (5 minutes) hot start, followed by 30 cycles of 94°C (1 minute), 60°C (1 minute), 72°C (1 minute), and 5 minutes at 72°C to ensure double-stranded cDNA. Isolated cDNA fragments were digested with PstI, subcloned into the multiple-cloning site of the pGem-4z plasmid (Promega), and transformed into XL1-blue high-efficiency Escherichia coli host cells (Stratagene). Differential expression of the clones of interest was verified by Northern blot hybridization and/or RT-PCR.
Sequence Analysis
All subcloned cDNA fragments were sequenced with the M13/pUC forward primer and the ABI Prism Ready Reaction Dideoxy Terminator Cycle Sequencing Kit (Perkin Elmer) following the manufacturer’s protocol. The sequencing reaction products were resolved on an ABI PRISM Automated DNA Sequencer (ABI 377, Applied Biosystems). The differentially expressed cDNA clones were compared with a non-redundant nucleotide sequence database that includes sequences from the Brookhaven Protein Data Bank, GenBank, GenBank updates, European Molecular Biology Laboratories, and updates with the BLAST and the FastA algorithm at the National Center for Biotechnology Information.21
Northern Blot Analysis
RNA (10 µg/lane) was separated by electrophoresis on a 1.2% agarose, 2.2 mol/L formaldehyde gels and transferred onto a nylon membrane (Amersham, Hybond N). Hybridization was performed by using QuickHyb hybridization solution (Stratagene) and 1 to 5x107 cpm of 32P-labeled PstI-cut fragments of the cloned rat cDNAs. Filters were washed at 65°C with increasing stringency with 2xSSC/0.1%SDS up to 0.1xSSC/0.1%SDS before exposure to x-ray film at -70°C. Filters were stripped and rehybridized to a probe for ribosomal RNA or a 0.8-kilobase PstI fragment for glyceraldehyde-3-phosphate dehydrogenase as a control for gel loading and transfer.
RT-PCR
First-strand synthesis (5 µg total RNA) was performed with the Superscript Preamplification Kit (Gibco BRL) by using Oligo-dT12–18 oligonucleotides. Quality of reverse-transcribed cDNA (absence of contamination with genomic DNA and equality of cDNA amount) was verified by RT-PCR by using intron spanning primers for ß-actin (Clontech). All PCR reactions were performed with standard conditions unless otherwise stated. The PCR temperature profile used was 94°C (5 minutes) hot start, followed by 30 cycles of 94°C (1 minute), 60°C (1 minute), 72°C (1 minute), and 5 minutes at 72°C. Optimal PCR conditions were established by determining the relationship between signal strength and number of PCR cycles for Zfhep (n=3 for each condition) and ß-actin, and saturation of PCR reaction was reached at more than 33 PCR cycles for ß-actin whereas Zfhep mRNA was detectable for the first time after 30 cycles (data not shown). Amplimers for the ß-actin control were verified for the correct size (1128 bp) to ensure quality of cDNA and lack of contamination with genomic DNA. Identity of the Zfhep PCR products was verified by hybridization to the original clone isolated from the differential mRNA display by using standard protocols for slot blots after RT-PCR. Autoradiographic signals were densitometrically quantified using NIH Image.
Oligonucleotides
Oligonucleotides used for RT-PCR cDNA were ß-actin sense: 4'-ATGGATGATGATATCGCCGCG-3', ß-actin antisense: 5'-CTAGAAGCATTTGCGGTGGACGATGGAGGGGCC-3', Zfhep sense: 5'-CACATTAAGTACCGCCATGAGA, and Zfhep antisense: 5'-GTTGTGCCATCCTGATCAACTA.
Statistics
All experiments were performed in at least 4 independent experiments from 3 individual isolations of CECs or individual subcultures of PC12w, respectively. Statistical analysis of differential gene expression was performed on the data from densitometric analysis by using one-way ANOVA followed by appropriate post hoc tests.
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Results |
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Differential mRNA Display
In the initial differential mRNA display, the gene expression in PC12w cells after stimulation with 50 ng/mL EGF was compared with cells stimulated with 50 ng/mL EGF plus Ang II (10-7 mol/L) at 1 and 6 hours. Quiescent cells served as controls to account for constitutive gene expression. The band pattern of the display after amplification of the respective cDNAs with the AzPstI-primer revealed 9 differentially amplified bands (200 to 450 bp), of which most were found to be different after 6 hours and three were verified with Northern blots. In addition to these, we isolated one fragment that was markedly expressed after 1 hour in the EGF group. This clone was 69% homologous to human insulin-like growth factor-2 (IGF-2) and showed a marked differential expression on the initial Northern blot with a size of
900 bp that is comparable to the reported size for IGF-2 (Figure 1). However, a differential expression of this clone was only observed between quiescent PC12w cells and EGF-treated cells, an effect that was not affected by Ang II. Sequence analysis of three of the clones differentially displayed in the 6-hour group revealed for clone 2: 100% identity to U52584 (rat Zfhep-1/2) in 224-bp overlap, for clone 3: 100% identity to D43778 (rat angiotensin AT2 receptor) in a 198-bp overlap, and for clone 4: 85% identity to Y00168 (rat fibroblast tropomyosin 4) in 135-bp overlap. The sequence of clone 2 (Zfhep-1/2) obtained from the differential mRNA display, as well as its relation to the known open reading frame, is presented in Figure 2. Northern blot analysis with the total RNA from the initial experiment for the differential display RT-PCR (DDRT-PCR) confirmed a differential mRNA expression for Zfhep after stimulation with EGF±Ang II at 1 and 6 hours after stimulation (Figure 1). Zfhep was induced after 1 hour and downregulated after 6 hours by Ang II. Clone 3 was identified as a partial sequence of the AT2 receptor. Northern blot analysis with the RNA from the initial experiment for the DDRT-PCR verified a differential expression of AT2 receptor mRNA in response to EGF and AT2 receptor stimulation with an 80-fold increase in AT2 mRNA levels by EGF at 1 hour, which was not affected by Ang II, and a 88-fold increase by EGF at 6 hours, which was reduced by 40% by Ang II (Figure 1).
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Differential Expression of Zfhep in PC12w Cells
Zfhep22 hybridized to a mRNA of approximately 3.4 to 3.5 kb, which is in accordance with the reported size for the homologous mRNA reported for Zfhep-1 (3403bp). The expression level of this clone on mRNA level was very low, but a regulation of the Zfhep mRNA was clearly detectable (Figure 1) with a rapid induction of Zfhep 1 hour after stimulation with EGF (50 ng/mL) ± Ang II (10-7 mol/L). Six hours after stimulation, Zfhep mRNA was still detectable in EGF-treated cells, but no longer present in EGF+Ang II–treated cells (Figure 1), thus confirming the initial mRNA display which revealed downregulation of this band by Ang II 6 hours after stimulation. This initial experiment indicated that Ang II may regulate growth factor–induced Zfhep expression in PC12w cells; however, it did not address whether Ang II via the AT2 receptor could directly regulate Zfhep in the absence of EGF. Therefore, subsequent experiments were performed in which quiescent PC12w cells were stimulated with Ang II (10-7 mol/L) in the presence or absence of the specific AT2 receptor antagonist PD123177 (10-6 mol/L) over a course of 1 to 24 hours. The AT1 receptor antagonist losartan was not tested in these experiments because PC12w cells in the passages used express exclusively AT2 receptors.19 As shown in Figure 3, no Zfhep mRNA expression was detected in quiescent PC12w cells. Treatment with Ang II resulted in a rapid induction of Zfhep mRNA expression, an effect that was abolished in the presence of PD123177 (Figure 3). Three hours after stimulation, Zfhep expression was no longer detectable throughout the course of the experiment.
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Because the basal expression level of Zfhep was very low and difficult to quantify using Northern blot analysis, we designed primers based on the published sequence and used semi-quantitative RT-PCR for subsequent experiments. These experiments were also performed to verify that the isolated clone was indeed Zfhep, because the PCR products were hybridized to the original clone isolated from the differential mRNA display by slot blot hybridization. In these experiments, a basal expression of Zfhep in quiescent PC12w cells was detectable by using RT-PCR. This expression was enhanced 1 hour and 3 hours after stimulation with Ang II (10-7 mol/L), an effect that was abolished in the presence of the AT2 receptor antagonist PD123177 (10-6 mol/L; Figure 3b).
Induction of Zfhep mRNA Expression in CEC by AT2-Receptor Stimulation
In a second set of experiments, we investigated the gene expression of Zfhep in rat CECs to evaluate the AT2 receptor–related gene expression in cells of vascular origin. In addition, these cells have been shown to express both Ang II receptors,3 allowing the study of a potential role of the AT1 receptor in Zfhep expression. The expression of AT1 and AT2 receptors was verified before the experiments by RT-PCR (data not shown). Similar to our experiments in PC12w cells we studied Ang II-induced effects over a course of 1 to 6 hours. In the experiments on quiescent CECs, 25 ng/mL bFGF served as a positive control inducing Zfhep up to 10-fold (Figure 4). Ang II (10-7 mol/L) did not induce any Zfhep mRNA when both receptor subtypes were accessible. However, after pretreatment with the selective AT1 receptor antagonist losartan (10-5 mol/L), stimulation with Ang II (10-7 mol/L) resulted in a marked expression of Zfhep mRNA, an effect that exceeded the effect induced by bFGF (27-fold); whereas in the presence of the AT2 receptor antagonist PD123177 (10-6 mol/L) no Zfhep mRNA expression was detectable (Figure 4). A similar expression pattern was observed in CECs stimulated with 25 ng/mL bFGF 16 hours before application of Ang II (10-7 mol/L) in the absence or presence of losartan (10-5 mol/L) and PD123177 (10-6 mol/L). This protocol is identical to the protocol applied when we investigated the growth-inhibiting properties of the AT2 receptor in CECs3 and was aimed to investigate whether the known antiproliferative effect of the AT2 receptor in these cells is accompanied by changes in the expression of Zfhep mRNA under identical experimental conditions. Sixteen hours after stimulation with bFGF, Zfhep mRNA levels were no longer induced in bFGF-treated cells compared with quiescent controls (Figure 5). Figure 5a shows representative results from RT-PCR in CECs; Figure 5b summarizes the data from 5 to 7 different experiments revealing that Zfhep is upregulated by Ang II only when the AT1 receptor is blocked by losartan. As observed in quiescent cells, Ang II alone did not markedly alter Zfhep mRNA expression in bFGF-treated cells, 1 and 3 hours after stimulation with Ang II. However, in the presence of the AT1 receptor antagonist losartan, a significant increase in Zfhep mRNA was observed up to 3 hours after stimulation (P<0.01 versus bFGF+Ang II), an effect that was reversed by the addition of the AT2 receptor antagonist PD123177 (P<0.01 versus bFGF+Ang II+PD123177 and P<0.05 versus bFGF+Ang II+losartan+PD123177). Stimulation of the AT1 receptor, by blockade of the AT2 receptor, did not affect Zfhep mRNA expression in the presence of bFGF (Figure 5a and 5b), nor did the addition of the selective antagonists (not significant versus bFGF). In addition, Northern blot analysis was used to verify AT2-mediated induction of Zfhep mRNA in CECs. Although the expression level of Zfhep was very low, and the bands after Northern hybridization were weak and induction of Zfhep was observed after AT2 stimulation (bFGF+Ang II+losartan) supporting the RT-PCR data. The AT2-receptor–mediated induction of Zfhep was again an early (1 hour) and transient phenomenon (data not shown).
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Discussion |
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Apart from its cardiovascular effects, the control of cellular proliferation, differentiation, and apoptosis appears to constitute major actions of Ang II. Recent studies implicate a differential role of both AT1 and AT2 receptors in these processes (for review, see de Gasparo5). Growth stimulatory effects induced by Ang II are usually associated with the stimulation of AT1 receptors,2,23 whereas AT2 receptors have been shown to inhibit the proliferation of various cell types in vitro3,19,24 and in vivo1,25 and to induce morphological differentiation in cells of neuronal origin.26 Furthermore the AT2 receptor may act in a pro-apoptotic manner9,27 and has been connected with regenerative processes after tissue injury;10,12 both by its biological activity and its clear upregulation during embryogenesis28 and after injury.11 Using differential mRNA display, we isolated differentially expressed genes after stimulation of AT2 receptors to further characterize downstream events of AT2 signaling and to identify potential genes that are involved in the AT2-induced antigrowth actions. From the initial nine differentially displayed PCR products after stimulation of PC12w cells with EGF±Ang II, three genes were successfully confirmed by Northern blot. Although each of the clones were of potential interest, we prioritized the characterization of Zfhep22, because it appeared to directly relate to the proposed function of the AT2 receptor. Zfhep belongs to the zfh family of Drosophila genes that are required for differentiation of the central nervous system and in the regulation of cell fate.29,30 In addition, studies on the vertebrate homologue, ZEB, have extended the findings to a role in myogenesis, skeletal development and early T-cell development.31,32
Our initial experimental design for differential mRNA display (EGF versus EGF+Ang II versus control at 1 and 6 hours) was chosen for two reasons: (1) to limit the number of potential genes of interest to a manageable level, and (2) to capture genes involved in the events preceding the growth-inhibiting and differentiation-promoting actions of the AT2 described in these cells.19 In the course of the characterization of Zfhep, we verified the involvement of AT2 receptor activation in Zfhep expression in the absence of EGF, conditions in which the AT2 receptor is known to induce differentiation of PC12w cells.19 In quiescent PC12w cells, Ang II transiently induced the expression of Zfhep, 1 and 3 hours after stimulation, an effect that was AT2-specific because it was abolished by the AT2 antagonist PD123177. Studies were then extended to CECs to determine whether the observed induction of Zfhep mRNA was specific to neuronal cells that undergo differentiation, or whether this induction can also be observed in cells of vascular origin. This was particularly interesting as PC12w cells exclusively express AT2 receptors whereas CECs express both AT1 and AT2 receptors and respond differently to Ang II depending on which receptor subtype is amenable.3 Our results confirm that the AT2 receptor induces Zfhep in CECs as well. However, an effect of Ang II on CECs was only detected when the AT1 receptors were blocked by losartan. The finding that simultaneous stimulation of both receptors by Ang II was ineffective on Zfhep expression suggests that the AT1 receptor does not stimulate Zfhep transcription but downregulates AT2-induced Zfhep mRNA expression. In all previous reports, the Ang II–mediated induction of transcription factors was attributed to the AT1 receptor,33,34 an effect that was unopposed by the AT2 receptor.35 To our knowledge, this is the first report on the induction of a transcription factor via the AT2 receptor that is opposed by the AT1 receptor.
In our opinion, Zfhep represents a suitable marker for the study of AT2 receptor activation and the subsequent elucidation of signaling events involved in the regulation of cell growth and differentiation. For example, a yeast-2-hybrid screen revealed an interaction between the third intracellular loop of the AT2 receptor and a member of the EGF receptor family, ErbB3, as part of a novel signaling mechanism for the AT2 receptor–mediated inhibition of cell growth.36 Our initial experiment for the differential mRNA display examined such an interaction, the influence of AT2 receptor activation on EGF-induced growth in PC12w, which ultimately led to the discovery of Zfhep. Therefore, Zfhep may represent an important link between early events in receptor-receptor–cross talk, such as AT2-ErbB3, and downstream target genes involved in cellular growth and differentiation. The question remains regarding the biological role of Zfhep within AT2-mediated effects. The presence of a homoeobox domain implies a putative action on differentiation genes. Recent studies showed that the mouse homologue ZEB down-modulates the activity of transcriptional activators involved in cell differentiation such as c-myb, NF-
B p65, MEF2C, ITF-1, and myoD.37 Since differentiation requires positive and negative regulation of transcriptional activators to ensure a proper temporospatial pattern of gene expression, it was postulated that ZEB is involved in the orchestration of differentiation events.37 Similarly, the AT2 receptor has been proposed to participate in events leading to cell differentiation and/or apoptosis.27,38 It is conceivable that, via the induction of Zfhep or its homologues, the AT2 receptor exerts a subtle role in the orchestration of differentiation during embryogenesis.
Interestingly, one of the clones "fished" in our differential mRNA display was the AT2 receptor itself and the Northern blot confirmed a concordant regulation of both, AT2 and Zfhep mRNA in PC12w cells. It furthermore confirmed a temporal transcriptional regulation of AT2 receptors by growth factors, in our experimental set-up EGF, with a subsequent down regulation, which is in accord with the observation by other investigators for fetal calf serum, transforming growth factor-beta and bFGF.39 The promoter of the rat AT2 receptor contains several putative consensus sequences, such as AP-1 and AP-3, as well as a MyoD and a Myc binding site.40 Unfortunately, the promoter sequence of Zfhep has not yet been characterized. Based on our current data, the concordant expression of Zfhep and AT2 mRNA may be a coincidence. However, considering the overall data on expression of both genes, with a predominant expression during embryogenesis and a later restriction to select tissues or processes that involve differentiation, it is conceivable that both genes are regulated through a joint pathway.
In summary, we have identified a gene that is induced after activation of AT2, but not AT1, receptors in cells of neuronal and vascular origin. This gene encodes for a transcription factor recognized for its role in the regulation of cellular differentiation and is expressed under experimental conditions where the AT2 receptor exerts its antiproliferative and differentiation-promoting effects. In conclusion, Zfhep not only represents a suitable marker for AT2 receptor activation, but it may also link AT2 signaling and downstream events involved in the proposed function of the AT2 receptor in development and regeneration.
Received September 8, 2001; accepted September 30, 2001.
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