Published online before print August 28, 2006, doi: 10.1161/01.HYP.0000240064.19301.1b
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
(Hypertension. 2006;48:564.)
© 2006 American Heart Association, Inc.
Original Articles |
Prorenin Induces Intracellular Signaling in Cardiomyocytes Independently of Angiotensin II
From the Departments of Pharmacology (J.J.S., I.M.G., A.H.J.D.) and Biochemistry (D.H.W.D., J.M.J.L.), Erasmus MC, Rotterdam, The Netherlands; the Center for Human and Clinical Genetics (P.A.C.t.H., J.T.d.D.), and the Leiden Genome Technology Center (J.T.d.D.), Leiden University Medical Center, Leiden, The Netherlands.
Correspondence to A.H. Jan Danser, Department of Pharmacology, Room EE1418b, Erasmus MC, Dr Molewaterplein 50, 3015 GE Rotterdam, The Netherlands. E-mail a.danser{at}erasmusmc.nl
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Tissue accumulation of circulating prorenin results in angiotensin generation, but could also, through binding to the recently cloned (pro)renin receptor, lead to angiotensin-independent effects, like p42/p44 mitogen-activated protein kinase (MAPK) activation and plasminogen-activator inhibitor (PAI)-1 release. Here we investigated whether prorenin exerts angiotensin-independent effects in neonatal rat cardiomyocytes. Polyclonal antibodies detected the (pro)renin receptor in these cells. Prorenin affected neither p42/p44 MAPK nor PAI-1. PAI-1 release did occur during coincubation with angiotensinogen, suggesting that this effect is angiotensin mediated. Prorenin concentration-dependently activated p38 MAPK and simultaneously phosphorylated HSP27. The latter phosphorylation was blocked by the p38 MAPK inhibitor SB203580. Rat microarray gene (n=4800) transcription profiling of myocytes stimulated with prorenin detected 260 regulated genes (P<0.001 versus control), among which genes downstream of p38 MAPK and HSP27 involved in actin filament dynamics and (cis-)regulated genes confined in blood pressure and diabetes QTL regions, like Syntaxin-7, were overrepresented. Quantitative real-time RT-PCR of 7 selected genes (Opg, Timp1, Best5, Hsp27, pro-Anp, Col3a1, and Hk2) revealed temporal regulation, with peak levels occurring after 4 hours of prorenin exposure. This regulation was not altered in the presence of the renin inhibitor aliskiren or the angiotensin II type 1 receptor antagonist eprosartan. Finally, pilot 2D proteomic differential display experiments revealed actin cytoskeleton changes in cardiomyocytes after 48 hours of prorenin stimulation. In conclusion, prorenin exerts angiotensin-independent effects in cardiomyocytes. Prorenin-induced stimulation of the p38 MAPK/HSP27 pathway, resulting in alterations in actin filament dynamics, may underlie the severe cardiac hypertrophy that has been described previously in rats with hepatic prorenin overexpression.
Key Words: p38 MAP kinase • actin • microarray • hypertrophy • HSP27
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Prorenin, the inactive precursor of renin, circulates in human plasma in excess of renin, at concentrations that are up to 100 times higher.1 The reasons for this are unknown, but an attractive concept is that circulating prorenin is taken up at tissue sites where it contributes, after its local activation to renin, to tissue angiotensin production. Evidence for this concept comes from studies in transgenic animals displaying (inducible) prorenin expression.2,3 Prorenin activation at tissue sites might involve proteolytic removal of its prosegment.4 Alternatively, activation could occur in a nonproteolytic manner, for instance, through binding to a receptor. Indeed, Ichihara et al5 have proposed recently that human prorenin has so-called gate and handle regions for nonproteolytic activation. According to this concept, the handle region interacts with a putative receptor, which subsequently leads to unfolding of the gate region from the renin molecule. In vivo treatment with a decoy peptide corresponding to the gate region reduced the renal content of angiotensin (Ang) I and II in streptozotocin-induced diabetic rats, thereby supporting, for the first time, tissue Ang production by endogenous prorenin.
Interestingly, Nguyen et al6 have cloned a (pro)renin receptor, which exactly fulfills the above description, because prorenin binding to this receptor allowed prorenin to become fully enzymatically active in a nonproteolytic manner. Unexpectedly, (pro)renin binding to this receptor in glomerular mesangial cells also induced angiotensin-independent effects, that is, an increase in DNA synthesis, activation of the mitogen-activated protein kinases (MAPKs) extracellular signal regulated kinase 1 (p44)/extracellular signal regulated kinase 2 (p42), and plasminogen-activator inhibitor (PAI)-1 release,6,7 thus leading the authors to suggest that prorenin acts as an agonist of this receptor.
Prorenin, a phosphomannosylated protein, also binds to mannose 6-phosphate/insulin-like growth factor II receptors (IGF2R). Binding to these receptors is followed by internalization, intracellular proteolytic cleavage to renin, and subsequent proteolytic/hydrolytic removal or clearance.4 Although prorenin binding to IGF2R did not result in Ang generation,8 it is still possible that prorenin, like other M6P-containing proteins, acts as an agonist of IGF2R.
In the present study, we set out to investigate the angiotensin-independent effects of prorenin in neonatal rat cardiomyocytes using a gene transcription profiling approach. Cardiomyocytes were chosen not only because they express IGF2R4 but also because transgenic rats expressing prorenin exclusively in the liver (resulting in a 400-fold rise in plasma prorenin) display severe cardiac hypertrophy in the absence of hypertension.2 We verified the expression of the recently cloned (pro)renin receptor in cardiomyocytes. Finally, we evaluated the previously reported, angiotensin-independent, effects of prorenin mediated through this receptor.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Cell Culture
All of the experiments were performed according to the regulations of the Animal Care Committee of the Erasmus MC in accordance with the Guiding Principles of the American Physiological Society. Primary cultures of neonatal Wistar rat (Harlan) cardiomyocytes were prepared as described before.4 Cells were serum deprived for 24 hours, rinsed 3 times with 1 mL of warm (37°C) PBS, and incubated for a maximum of 48 hours at 37°C with 1 mL of serum-free medium, supplemented with 1% BSA, and containing 100 U/L (
2 nmol/L) of recombinant human prorenin4 and/or 150 nmol/L of human angiotensinogen (Sigma) in the presence or absence of M6P, eprosartan, PD123319, aliskiren (a gift of Novartis, Basel, Switzerland) or SB203580 (Sigma) to block IGF2R, angiotensin II type 1 (AT1) receptors, angiotensin II type 2 (AT2) receptors, renin, and p38 MAPK, respectively. For comparison, experiments with 1000 U/L of prorenin, 100 U/L of nonglycosylated prorenin,9 100 nmol/L of Ang II (Bachem), or 100 nmol/L of endothelin-1 (ET-1; Sigma) were also performed. Cells incubated without (ant) agonists served as control. At the end of each experiment, cells and medium were collected and stored at –70°C.
(Pro)renin Receptor Expression
Cardiomyocytes were lysed in Laemmli loading buffer (LLB). Skeleton protein–deprived fractions were obtained by centrifugation at 7000g for 30 minutes at 4°C. Fractions were boiled for 3 minutes, separated (20 µg) by SDS-PAGE (12%), and blotted to polyvinylidene fluoride membranes (Immunblot, Biorad). Equal protein distribution was assessed by Ponceau Red staining. Next, membranes were incubated overnight at 4°C with antibody SE3148 (a gift of Dr G. Nguyen, Collège de France, Paris, France), diluted 1:7500 in 150 mmol/L of NaCl buffered with 10 mmol/L of Tris and containing 0.1% Tween 20 (TTBS) and 0.5% milk powder after blocking nonspecific binding using the same buffer for 1 hour at room temperature. Receptor signal was visualized using a horseradish peroxidase–conjugated anti-rabbit antibody, SuperSignal WestFemto reagent (Pierce), and Hyperfilm ECL (Amersham Biosciences). To localize the receptor, cardiomyocytes were grown on glass cover slips at 3x104 cells/cm2, rinsed twice with cold PBS, fixed with 3% paraformaldehyde, permeabilized using 0.3% Triton X-100 in PBS, and blocked with 1% BSA in 0.1% Tween 20 containing PBS. Cells were then exposed to antibody SE3148 (1:500 in PBS with 0.1% Tween 20) for 1 hour at 37°C followed by overnight incubation at 4°C and subsequently incubated with biotinylated goat–anti-rabbit antibody (1:100) for 30 minutes at room temperature and tetramethylrhodamine B isothiocyanate-streptavidin (1:40) for 15 minutes at room temperature, both in PBS with Tween 20. Phalloidin–fluorescein isothiocyanate (15 ng/mL in PBS, 4 hours) was applied to stain actin filaments. Images were obtained with a Zeiss LSM-510 NLO confocal microscope system.
Biochemical Measurements
PAI-1 was measured with a sandwich ELISA using monoclonal antibodies specific for rat PAI-1 (a gift of P. Declerck, Katholieke Universiteit Leuven, Leuven, Belgium).10 MAPK activation was assessed by Western blotting, using cells lysates (20 µg) in LLB and antibodies specific for total and phosphorylated p42, p44, or p38 MAPK (Cell Signaling, 1:1000 in TTBS containing 75 mmol/L of NaCl and 0.5% milk powder for 4 hours at 20°C). Iodinated secondary antibodies (Amersham, 1:5000) were detected using a phosphor imager system (Biorad).
HSP27 phosphorylation was assessed by Western blotting using cell lysates (20 µg) in LLB, phospho-HSP27-Ser82 antibody (Cell Signaling, 1:1000 in TTBS containing 150 mmol/L of NaCl and 0.5% milk powder overnight at 4°C), and West Femto reagents.11 Quantification was done by densitometry with local background subtraction.
Gene Transcription Analysis
Cardiomyocytes were lysed using Tri-Reagent (Sigma-Aldrich). Individual purified RNAs were checked using Agilent Laboratory-on-Chip analysis (Agilent Bioanalyzer 2100) and UV spectrum analysis, equimolar pooled, amplified (Ambion), and Cye3 or Cye5 labeled, as described.12 Rat gene microarray slides representing Compugen/Laboratory-on-web 4.8-K oligonucleotide library in triplo were hybridized in a dye-swap loop protocol. Spot intensity measurement and global background (mean intensity plus 2xSD of 750 buffer-only spots) subtraction was performed within GenepixPro. Data files were uploaded in Rosetta Resolver for normalization, intensity ratio calculation, and confidence call estimation per gene or extracted with MS Excel, followed by variance stabilized normalization (VSN) in R and SAM analysis per individual spot.13,14 Original data files are accessible at National Center for Biotechnology Information (NCBI) Gene Expression Omnibus through Acc.ID GSE4340. Gene network building was performed using Ingenuity (www.ingenuity.com) software. For gene annotations, the National Institutes of Health Rat Genome Database Portal (http://rgd.mcw.edu/), NCBI Entrez (www.ncbi.nlm.nih.gov/gquery), and iHOP (www.pdg.cnb.uam.es/UniPub/iHOP) were used.
Quantitative Real-Time RT-PCR
Transcription of selected genes was quantified using intron-spanning assays on an ABI 7700 (Applied Biosystems Inc) using exon junction–specific probes15 or SYBR green chemistry for Col3a1 (forward: 5'AGGTCCTGCGGGTAACAST, reverse: 5'ACTTTCACCCTTGAYACCCTG).16 Superscript (0.2 U/mL), random hexamers (2.5 µmol/L), and RNAsin (0.5 U/mL; Promega) were used to transcribe 1 µg of RNA to cDNA in 30 µL for 60 minutes at 42°C. cDNAs were stored at –70°C and diluted 1:5 in H2O, and PCR reactions were performed with 5 µL of diluted cDNA in 25-µL reactions. Expression level was determined by threshold cycle number (CT) and, using the delta delta CT method, normalized against GAPDH and compared relative to the control in concordance with the manufacturer’s instructions.15
Statistical Analysis
Data are expressed as mean±SEM. Rosetta Resolver and Graphpad Prism were used for statistical analysis. P<0.05 was considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
(Pro)renin Receptor Expression
Western hybridization revealed (pro)renin receptor expression in the cytosol/microsome fraction of cardiomyocytes (Figure 1). Expression in the cytoskeleton fraction was close to the detection limit. Expression was also present in adult rat heart and brain homogenates. The cardiomyocyte cytoskeleton fraction contained a second band, which was not further investigated because of its low expression. Confocal microscopy results are in agreement with low plasma and high vesicular membrane staining of the receptor (Figure 1). Staining was absent after deletion of the primary antibody (data not shown).
|
PAI-1 Secretion and MAPK Activation
Prorenin plus angiotensinogen increased PAI-1 secretion to the same degree as Ang II (n=7 for all; Figure 2A), and this effect was blocked by 1 µmol/L of eprosartan but not 1 µmol/L of PD123319 or 10 mmol/L of M6P. When added separately, none of the receptor blockers (data not shown), prorenin, or angiotensinogen affected PAI-1 release significantly.
|
), and p44 MAPK (squares) activation (mean±SEM of 5 experiments) in cardiomyocytes by prorenin and ET-1. Bottom, representative experiment for p38 MAPK. *P<0.05 vs control (ie, cells without prorenin or ET-1).Prorenin (100 U/L) increased p38 MAPK but not p42 MAPK or p44 MAPK phosphorylation (n=5; Figure 2B). Peak p38 MAPK phosphorylation levels were reached after 45 minutes. ET-1 (n=5) activated all of the MAPKs at t=15 minutes. Phosphorylation of p38 MAPK at 1000 U/L of prorenin was 1.9±0.3 times (n=3) higher (P=0.03) than at 100 U/L, and nonglycosylated prorenin (100 U/L) increased p38 MAPK phosphorylation to the same degree as wild-type prorenin (to 201±23% of control; n=3).
Gene Expression Profiling and Validation
RNA was isolated from 5 separate experiments after 4 hours of stimulation with prorenin, Ang II, or vehicle (control). To obtain estimates of differential expression with equal precision between all of the samples (prorenin versus control, Ang II versus control, and prorenin versus Ang II), all of the possible sample pairs were cohybridized to the same array and dye replicated. Image analysis, filtering for genes with no detectable expression, and processing of background-corrected intensities with the Rosetta Resolver error model resulted in 4619 genes being amenable for comparison in all 3 combinations. The results of the pairwise comparisons (P<0.001) are given in Figure 3A and supplemental Table I (available online at http://hyper.ahajournals.org). In the prorenin versus control, Ang II versus control, and prorenin versus Ang II comparisons, respectively, 260, 215, and 177 genes were differentially expressed. Of these genes, 43%, 41%, and 12% were upregulated and displayed an absolute fold change of 
1.3. Comparable results were obtained when the data were analyzed with a different normalization algorithm (VSN)14 followed by SAM analysis13 (data not shown). Histogram analysis of the expression ratios (Figure 3B) revealed a more pronounced increase in number and activation of prorenin-responding genes as compared with Ang II–responding genes (Mann–Whitney test, P=0.0002).
|
, total number of genes for each comparison. B, Fold change distribution profile of the prorenin- (
) and angiotensin II–regulated (
) genes (P=0.0002). C, Quantitative real-time RT-PCR measurements (log2-ratio vs control; mean±SEM) in 5 individual mRNA samples obtained from cardiomyocytes stimulated with prorenin (To obtain a more robust set of prorenin-specific genes, the data were also analyzed by 1-way ANOVA (P<0.01). This resulted in 171 genes displaying differential expression between the 3 conditions, of which 91, 23, and 36 overlapped with the 3 above pairwise comparisons, respectively. The Table shows the 28 genes that were also not regulated by Ang II, that is, the truly "prorenin-specific" genes.
|
Seven genes (Opg, Timp1, Best5, Hsp27, pro-Anp [Nppa], Col3a1, and Hk2) were selected for further validation of the microarray data. Selection was based on magnitude of induction (eg, Best5) and/or cardiovascular relevance (eg, Timp1). Two of these genes were prorenin specific (Hsp27 and Hk2). Quantitative real-time RT-PCR on individual mRNAs (Figure 3C) and RNA pools (r=0.72; P=0.004; Figure 3D) confirmed the microarray results for these 7 genes. Because of large interindividual variation for Best5, the prorenin-induced effects on this gene were not significant when based on measurements in individual samples. Figure 4A shows the time course of gene regulation of the 7 selected genes during stimulation with prorenin. In most cases, the peak levels observed after 4 hours had returned to baseline after 24 hours.
|
To investigate the Ang II dependency of the effect of prorenin, the 4-hour incubation experiments with prorenin were repeated in the presence of 1 µmol/L of eprosartan (n=3) or 100 nmol/L of aliskiren (n=5). Neither drug blocked the effects of prorenin on the 7 genes (Figure 4B) nor did these drugs exert effects when added alone (data not shown).
Pathway Analysis
Uploading the 91 prorenin-regulated genes (resulting from the overlap between the pairwise comparison and the ANOVA) in Ingenuity allowed the construction of a backbone network of 140 genes (supplemental Figure I), derived by merging 4 main clusters. Of these genes, 58 were present on the array, and 51 of these 58 genes were affected by prorenin. Including all 187 genes that were regulated by prorenin but not Ang II according to the pairwise comparison (Figure 3A) did not result in a larger network but increased the number of small gene clusters (containing <4 genes; data not shown). Mapping of prorenin-regulated genes into functional groups in Ingenuity revealed the involvement of 17 genes in "Cellular Assembly and Organization," 5 in "Cellular Movement," 17 in "Nervous System Development and Function," and 7 in "Protein Trafficking" (P<0.001, exact Fisher test; supplemental Table II).
Transcription Factor Binding-Site Motif Frequency Distribution Analysis and Identification
Data were uploaded in the TELIS database (www.telis.ucla.edu)17 to find common transcription factor binding-site motifs (TFBMs) among the 187 of 260 genes that were regulated by prorenin but not Ang II. Using stringent criteria (NCBI_Refseq [Fall 2003], 300 nucleotide rat promotor sequences, homology stringency 0.95; P<0.005; false discovery rate 20%), the following TFBMs (representing 28 different genes, supplemental Table I) were overrepresented: ELK1_02 (TFBM for Arfrp1, Cdc10, Crot, Ddp2, Nap65, Nopp140, Prsc1, Rnp24, SRP54, Smn, Tkt, and Tsnax), CETS1P54_01 (Arfrp1, Cdc10, Ddp2, Ephx2, Fgg, Kcne1, Nap65, Nopp140, Pls3, Prsc1, SRP54, Slc25a4, Svs5, and Tsnax), NRF2_01 (Cdc10, Ddp2, Nap65, Nopp140, SRP54, and Tsnax), and AP1_Q2 (Crry, Csrp3, Hbp1, Hk2, Kcne1, Neo1, Omd, RAMP4, Stx7, Sybl1, and Timm10). Six of these 28 genes were also present in the main prorenin Ingenuity network. Constructing an Ingenuity network on the basis of the 28 TFBM genes yielded 19 genes that overlapped with the main prorenin Ingenuity network, 8 of which were present on the array and prorenin regulated (Csrp3, Lgmn, Pls3, Ptgds, Stx7, Sybl1, Tkt, and Vdac1).
Genes Confined to Cardiovascular QTL Regions
Ninety-six genes of the Compugen 4.8-K oligonucleotide library were positioned within cardiovascular QTL regions as retrieved from the TIGR Rat Gene Index (www.tigr.org/tdb/tgi/resourcerer/rat_qtl.shtml). Prorenin regulated 13 of these 96 independent genes (P<0.0001, exact Fisher test; supplemental Table I), including Synthaxin-7 (Stx7, see TFBM analysis and supplemental Figure I) and Arginase-1 (Arg1) on rat chromosome 1 and the actin cytoskeleton interacting genes Pdlim3/Actn2lp and Argbp2 on rat chromosome 16 (see below).
p38 MAPK Pathway
Activation of p38 MAPK has been reported to influence actin filament dynamics via MAPK-activated protein kinase 2 (MK2) and HSP27 activation.18 The prorenin-induced increases in mRNAs of cytokines that are known to be stimulated by MK2 (Il-1a, Il-1b, Mip3
/Scya20, and Ccl3/Mip1/Scya3)19,20 support the activation of this pathway by prorenin. HSP27 (Ser-82) phosphorylation measurements confirm prorenin-induced HSP27 activation in cardiomyocytes (Figure 5A). HSP27 phosphorylation peaked at 45 minutes (n=3, data not shown), occurred in a concentration-dependent manner, and was blocked by SB203580. Finally, 2D proteomic differential display experiments, performed on cardiomyocytes after 48 hours of prorenin stimulation, reveal 2D positional shifts and up/downregulation of, among others, actin protein spots. Figure 5B shows the identification of one of the actin protein spots selected for confirmation of identity. Alterations in actin filament dynamics were also reflected at the mRNA level, as 29% and 40% of genes with annotations containing "actin" or "LIM" (eg, Csrp3/Mlp) were regulated by prorenin (P<0.0001, Fisher’s exact test). LIM (Pfam-PF00412) was incorporated in this analysis, because LIM-domain containing proteins are known to interact with actin.21 The prorenin-induced upregulation of St2 ('c-FOS_responsive_gene-1'/Il1rl1) corroborates c-Fos activation by p38 MAPK.22
|
Renin–Ang System Genes and Ang II–Regulated Genes
Prorenin did not regulate renin–Ang system genes (including the IGF2R). In support of PAI-1 antigen release results, Pai1 mRNA levels were stimulated by Ang II but not prorenin. Ang II also regulated interleukin 6, atrial natriuretic peptide, and the adrenomedullin-related genes receptor activity- modifying protein-3 (Ramp3) and adrenomedullin precursor (Adm) in full agreement with previous investigations in cardiomyocytes.23–25
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
This study shows that recombinant human prorenin induces gene transcription in neonatal rat cardiomyocytes independently of Ang II generation. Human prorenin–induced Ang generation in these cells is highly unlikely, not only because of the species specificity of the renin–angiotensinogen reaction26 but also because our prorenin preparation contains very little (<2%) renin activity.4 Furthermore, angiotensinogen protein expression, if occurring in neonatal rat cardiomyocytes, is low.26 Indeed, in a previous study, we were unable to detect Ang generation by these cells after the addition of human prorenin to the medium.8 Finally, neither the renin inhibitor aliskiren nor the AT1 receptor antagonist eprosartan blocked the transcriptional effects of prorenin, thereby confirming that neither renin activity nor AT1 receptor stimulation are required for these effects.
If not involving Ang II, alternative mechanisms must be provided to explain the prorenin-induced regulation of gene transcription. Recent investigations support the existence of 2 prorenin-binding receptors coupling directly to second messenger systems: IGF2R, which serve as G protein–coupled receptors when exposed to M6P-containing proteins like prorenin,27 and (pro)renin receptors, which activate p42/p44 MAPK and release PAI-1 after exposure to (pro)renin.6,7 IGF2Rs are abundantly present in neonatal rat cardiomyocytes.4 The present study now shows that cardiomyocytes also express the (pro)renin receptor, although at low levels. Expression seemed to occur largely intracellularly. Although this contrasts with the cell surface location that was originally proposed in mesangial cells, it is consistent with the fact that the C-terminal tail of the (pro)renin receptor is identical to the M8-9/Atp6ap2 protein, which associates with the vacuolar H+-ATPase (V-ATPase) complex.28 The (pro)renin receptor antibody used in the present study (SE3148) was generated with an immunization peptide that overlapped the M8–9 sequence.6 V-ATPases maintain pH gradients between intracellular compartments and the cytoplasm by proton secretion,28 and, thus, prorenin might exert effects by interfering with this process.
Prorenin application to cardiomyocytes did not result in detectable p42/p44 MAPK activation or PAI-1 release as in human mesangial cells.6,7 Thus, either the expression of (pro)renin receptors in cardiomyocytes is too low, or their (intracellular) location does not allow such effects. PAI-1 release did occur when applying prorenin in combination with human angiotensinogen. Eprosartan, but not PD123319 or M6P, blocked this effect. Thus, in cardiomyocytes, unlike mesangial cells, PAI-1 release is entirely dependent on Ang generation and subsequent AT1 receptor activation. It does not require IGF2R. The Ang II–induced upregulation of Pai-1 expression complements this view.
Independently of Ang II generation (ie, in the absence of angiotensinogen), prorenin was found to activate p38 MAPK in cardiomyocytes. This effect occurred in a concentration-dependent manner and could be mimicked by nonglycosylated prorenin. The latter suggests that it does not involve IGF2R, thereby excluding a scenario in which the stimulation of intracellular (pro)renin receptors depends on IGF2R-mediated internalization of prorenin.29 Consequently, it seems that prorenin activates the p38 MAPK pathway in an IGF2R-independent manner, for instance, through direct stimulation of (pro)renin receptors.
Gene transcription profiling studies support and extend this view. Not only did these studies confirm prorenin-induced activation of the p38 MAPK pathway, evidenced by the stimulation of its downstream (MK2- and c-FOS–dependent) targets HSP27, Il-1a, Il-1b, Scya20, Ccl3/Mip1/Scya3, and St2,19,20,22 but they also reveal that prorenin regulates genes confined to cardiovascular QTL regions that are not necessarily linked to p38 MAPK activation. Regulation occurred in a time-dependent manner, with peak changes obtained after 4 hours of prorenin exposure. Overrepresentation of genes containing binding site motifs for the transcription factors Elk1, Ets1, Ap1, and Nrf2 (known effectors of the p38 MAPK pathway)30–32 further corroborates prorenin-induced p38 MAPK activation. It should be noted that Ang II also activates p38 MAPK in neonatal rat cardiomyocytes.33 This explains, at least in part, the overlap between prorenin- and Ang II–induced genes.
Renin–Ang system gene regulation was not observed after prorenin exposure, in agreement with the concept that the prorenin-induced effects occur in an Ang II–independent manner. Prorenin-induced p38 MAPK activation coincided with HSP27 phosphorylation, and the latter phenomenon was blocked by the p38 MAPK inhibitor SB203580. HSP27, through its regulation of actin filament dynamics, is believed to be involved in maintaining the integrity of cell architecture, growth, motility, survival, and death.34 Proteome differential display experiments performed on cardiomyocytes after 48 hours of prorenin stimulation support the downstream effects of HSP27 on the actin cytoskeleton, as does the overrepresentation of actin-related genes regulated by prorenin, like the LIM domain containing Csrp3/Mlp. The p38 MAPK-MK2 signaling pathway, involving both HSP27 and LIM-kinase 1, has also been linked to VEGF-induced actin reorganization in endothelial cells.21 Furthermore, in view of the observation that the (pro)renin receptor equals a subunit of the V-ATPase complex,28 V-ATPase transport might be responsible for the reorganization of actin cytoskeleton.35 In fact, V-ATPase-induced acidosis is sufficient to activate p38 MAPK and HSP27.36,37
Perspectives
The current data support prorenin-induced intracellular signaling in cardiomyocytes. This effect occurred independently of Ang II and IGF2R. The exact mechanism underlying this phenomenon is not yet known. One possibility is that it involves the recently discovered (pro)renin receptor. Our data suggest that prorenin directly affects cardiac growth and development, thereby providing an explanation for the Ang II- and blood pressure-independent cardiovascular damage observed in rats overexpressing prorenin.2 Consequently, the rise in prorenin in diabetes, particularly in subjects with microvascular complications,1 might be of functional relevance. Combined with the recent concept that prorenin becomes activated when bound to the (pro)renin receptor,5,6 a new class of drugs might emerge, that is, (pro)renin receptor blockers, which prevent both Ang generation at tissue sites and prorenin-induced, angiotensin-independent effects.
|
Acknowledgments |
|---|
Source of Funding
This study was supported by the Dutch Kidney Foundation, grant NSN C03.2042.
Disclosures
A.H.J.D. is the recipient of a Novartis Aliskiren Grant (significant) and is a member of the Novartis Aliskiren Advisory Board (modest). The remaining authors report no conflicts.
Received May 17, 2006; first decision June 5, 2006; accepted July 27, 2006.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
- Danser AHJ, Deinum J. Renin, prorenin and the putative (pro)renin receptor. Hypertension. 2005; 46: 1069–1076.
[Free Full Text] - Véniant M, Ménard J, Bruneval P, Morley S, Gonzales MF, Mullins JJ. Vascular damage without hypertension in transgenic rats expressing prorenin exclusively in the liver. J Clin Invest. 1996; 98: 1966–1970.[Medline] [Order article via Infotrieve]
- Prescott G, Silversides DW, Reudelhuber TL. Tissue activity of circulating prorenin. Am J Hypertens. 2002; 15: 280–285.[CrossRef][Medline] [Order article via Infotrieve]
- Saris JJ, Derkx FHM, de Bruin RJA, Dekkers DHW, Lamers JMJ, Saxena PR, Schalekamp MADH, Danser AHJ. High-affinity prorenin binding to cardiac man-6-P/IGF-II receptors precedes proteolytic activation to renin. Am J Physiol. 2001; 280: H1706–H1715.
- Ichihara A, Hayashi M, Kaneshiro Y, Suzuki F, Nakagawa T, Tada Y, Koura Y, Nishiyama A, Okada H, Uddin MN, Nabi AH, Ishida Y, Inagami T, Saruta T. Inhibition of diabetic nephropathy by a decoy peptide corresponding to the "handle" region for nonproteolytic activation of prorenin. J Clin Invest. 2004; 114: 1128–1135.[CrossRef][Medline] [Order article via Infotrieve]
- Nguyen G, Delarue F, Burcklé C, Bouzhir L, Giller T, Sraer J-D. Pivotal role of the renin/prorenin receptor in angiotensin II production and cellular responses to renin. J Clin Invest. 2002; 109: 1417–1427.[CrossRef][Medline] [Order article via Infotrieve]
- Nguyen G, Delarue F, Berrou J, Rondeau E, Sraer JD. Specific receptor binding of renin on human mesangial cells in culture increases plasminogen activator inhibitor-1 antigen. Kidney Int. 1996; 50: 1897–1903.[Medline] [Order article via Infotrieve]
- Saris JJ, van den Eijnden MMED, Lamers JMJ, Saxena PR, Schalekamp MADH, Danser AHJ. Prorenin-induced myocyte proliferation: no role for intracellular angiotensin II. Hypertension. 2002; 39: 573–577.
[Abstract/Free Full Text] - van den Eijnden MMED, Saris JJ, de Bruin RJA, de Wit E, Sluiter W, Reudelhuber TL, Schalekamp MADH, Derkx FHM, Danser AHJ. Prorenin accumulation and activation in human endothelial cells. Importance of mannose 6-phosphate receptors. Arterioscler Thromb Vasc Biol. 2001; 21: 911–916.
[Abstract/Free Full Text] - Ngo TH, Verheyen S, Knockaert I, Declerck PJ. Monoclonal antibody-based immunoassays for the specific quantitation of rat PAI-1 antigen and activity in biological samples. Thromb Haemost. 1998; 79: 808–812.[Medline] [Order article via Infotrieve]
- Faber MJ, Dalinghaus M, Lankhuizen IM, Bezstarosti K, Dekkers DHW, Duncker DJ, Helbing WA, Lamers JMJ. Proteomic changes in the pressure overloaded right ventricle after 6 weeks in young rats: Correlations with the degree of hypertrophy. Proteomics. 2005; 5: 2519–2530.[CrossRef][Medline] [Order article via Infotrieve]
- ’t Hoen PA, de Kort F, van Ommen GJ, den Dunnen JT. Fluorescent labelling of cRNA for microarray applications. Nucleic Acids Res. 2003; 31: e20.
[Abstract/Free Full Text] - Tusher VG, Tibshirani R, Chu G. Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A. 2001; 98: 5116–5121.
[Abstract/Free Full Text] - Huber W, von Heydebreck A, Sultmann H, Poustka A, Vingron M. Variance stabilization applied to microarray data calibration and to the quantification of differential expression. Bioinformatics. 2002; 18: S96–S104.[Abstract]
- Lemos FB, IJzermans JN, Zondervan PE, Peeters AM, van den Engel S, Mol WM, Weimar W, Baan CC. Differential expression of heme oxygenase-1 and vascular endothelial growth factor in cadaveric and living donor kidneys after ischemia-reperfusion. J Am Soc Nephrol. 2003; 14: 3278–3287.
[Abstract/Free Full Text] - Wang X, Seed B. A PCR primer bank for quantitative gene expression analysis. Nucleic Acids Res. 2003; 31: e154.
[Abstract/Free Full Text] - Cole SW, Yan W, Galic Z, Arevalo J, Zack JA. Expression-based monitoring of transcription factor activity: The TELiS database. Bioinformatics. 2005; 21: 803–810.
[Abstract/Free Full Text] - Guay J, Lambert H, Gingras-Breton G, Lavoie J, Huot J, Landry J. Regulation of actin filament dynamics by p38 map kinase-mediated phosphorylation of heat shock protein 27. J Cell Sci. 1997; 110: 357–368.[Abstract]
- Kotlyarov A, Gaestel M. Is MK2 (mitogen-activated protein kinase-activated protein kinase 2) the key for understanding post-transcriptional regulation of gene expression? Biochem Soc Trans. 2002; 30: 959–963.[CrossRef][Medline] [Order article via Infotrieve]
- Clark A. Post-transcriptional regulation of pro-inflammatory gene expression. Arthritis Res. 2000; 2: 172–174.[CrossRef][Medline] [Order article via Infotrieve]
- Kobayashi M, Nishita M, Mishima T, Ohashi K, Mizuno K. MAPKAPK-2-mediated LIM-kinase activation is critical for VEGF-induced actin remodeling and cell migration. EMBO J. 2006; 25: 713–726.[CrossRef][Medline] [Order article via Infotrieve]
- Weinberg EO, Shimpo M, Hurwitz S, Tominaga S-i, Rouleau J-L, Lee RT. Identification of serum soluble ST2 receptor as a novel heart failure biomarker. Circulation. 2003; 107: 721–726.
[Abstract/Free Full Text] - Sano M, Fukuda K, Kodama H, Takahashi T, Kato T, Hakuno D, Sato T, Manabe T, Tahara S, Ogawa S. Autocrine/paracrine secretion of IL-6 family cytokines causes angiotensin II-induced delayed STAT3 activation. Biochem Biophys Res Commun. 2000; 269: 798–802.[CrossRef][Medline] [Order article via Infotrieve]
- Mishima K, Kato J, Kuwasako K, Imamura T, Kitamura K, Eto T. Angiotensin II modulates gene expression of adrenomedullin receptor components in rat cardiomyocytes. Life Sci. 2003; 73: 1629–1635.[CrossRef][Medline] [Order article via Infotrieve]
- Tsuruda T, Kato J, Kitamura K, Imamura T, Koiwaya Y, Kangawa K, Komuro I, Yazaki Y, Eto T. Enhanced adrenomedullin production by mechanical stretching in cultured rat cardiomyocytes. Hypertension. 2000; 35: 1210–1214.
[Abstract/Free Full Text] - van Kesteren CAM, Saris JJ, Dekkers DHW, Lamers JMJ, Saxena PR, Schalekamp MADH, Danser AHJ. Cultured neonatal rat cardiac myocytes and fibroblasts do not synthesize renin or angiotensinogen: evidence for stretch-induced cardiomyocyte hypertrophy independent of angiotensin II. Cardiovasc Res. 1999; 43: 148–156.
[Abstract/Free Full Text] - Hawkes C, Jhamandas JH, Harris KH, Fu W, MacDonald RG, Kar S. Single transmembrane domain insulin-like growth factor-II/mannose-6-phosphate receptor regulates central cholinergic function by activating a G-protein-sensitive, protein kinase C-dependent pathway. J Neurosci. 2006; 26: 585–596.
[Abstract/Free Full Text] - Ludwig J, Kerscher S, Brandt U, Pfeiffer K, Getlawi F, Apps DK, Schägger H. Identification and characterization of a novel 9.2-kDa membrane sector-associated protein of vacuolar proton-ATPase from chromaffin granules. J Biol Chem. 1998; 273: 10939–10947.
[Abstract/Free Full Text] - Catanzaro DF. Physiological relevance of renin/prorenin binding and uptake. Hypertens Res. 2005; 28: 97–105.[Medline] [Order article via Infotrieve]
- Wasylyk C, Bradford AP, Gutierrez-Hartmann A, Wasylyk B. Conserved mechanisms of Ras regulation of evolutionary related transcription factors, Ets1 and Pointed P2. Oncogene. 1997; 14: 899–913.[CrossRef][Medline] [Order article via Infotrieve]
- Anwar AA, Li FY, Leake DS, Ishii T, Mann GE, Siow RC. Induction of heme oxygenase 1 by moderately oxidized low-density lipoproteins in human vascular smooth muscle cells: role of mitogen-activated protein kinases and Nrf2. Free Radic Biol Med. 2005; 39: 227–236.[CrossRef][Medline] [Order article via Infotrieve]
- Tanos T, Marinissen MJ, Leskow FC, Hochbaum D, Martinetto H, Gutkind JS, Coso OA. Phosphorylation of c-Fos by members of the p38 MAPK family. Role in the AP-1 response to UV light. J Biol Chem. 2005; 280: 18842–18852.
[Abstract/Free Full Text] - Nishida M, Tanabe S, Maruyama Y, Mangmool S, Urayama K, Nagamatsu Y, Takagahara S, Turner JH, Kozasa T, Kobayashi H, Sato Y, Kawanishi T, Inoue R, Nagao T, Kurose H. G alpha 12/13- and reactive oxygen species-dependent activation of c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase by angiotensin receptor stimulation in rat neonatal cardiomyocytes. J Biol Chem. 2005; 280: 18434–18441.
[Abstract/Free Full Text] - Burridge K, Wennerberg K. Rho and Rac take center stage. Cell. 2004; 116: 167–179.[CrossRef][Medline] [Order article via Infotrieve]
- Vitavska O, Merzendorfer H, Wieczorek H. The V-ATPase subunit C binds to polymeric F-actin as well as to monomeric G-actin and induces cross-linking of actin filaments. J Biol Chem. 2005; 280: 1070–1076.
[Abstract/Free Full Text] - Zheng M, Hou R, Xiao RP. Acidosis-induced p38 MAPK activation and its implication in regulation of cardiac contractility. Acta Pharmacol Sin. 2004; 25: 1299–1305.[Medline] [Order article via Infotrieve]
- Kan H, Birkle D, Jain AC, Failinger C, Xie S, Finkel MS. p38 MAP kinase inhibitor reverses stress-induced cardiac myocyte dysfunction. J Appl Physiol. 2005; 98: 77–82.
[Abstract/Free Full Text]
Related Article:
- Prorenin and Its Ancient Receptor
- Céline Burcklé and Michael Bader
Hypertension 2006 48: 549-551.[Full Text] [PDF]
This article has been cited by other articles:
 |
|
 |
|
  M. J. Brown Aliskiren Circulation, August 12, 2008; 118(7): 773 - 784. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. W. Batenburg, R. J.A. de Bruin, J. M.G. van Gool, D. N. Muller, M. Bader, G. Nguyen, and A. H. J. Danser Aliskiren-Binding Increases the Half Life of Renin and Prorenin in Rat Aortic Vascular Smooth Muscle Cells Arterioscler. Thromb. Vasc. Biol., June 1, 2008; 28(6): 1151 - 1157. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. J. Kang, I. Toma, A. Sipos, E. J. Meer, S. L. Vargas, and J. Peti-Peterdi The Collecting Duct Is the Major Source of Prorenin in Diabetes Hypertension, June 1, 2008; 51(6): 1597 - 1604. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Nguyen and A. H. J. Danser Prorenin and (pro)renin receptor: a review of available data from in vitro studies and experimental models in rodents Exp Physiol, May 1, 2008; 93(5): 557 - 563. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. J. Campbell Critical Review of Prorenin and (Pro)renin Receptor Research Hypertension, May 1, 2008; 51(5): 1259 - 1264. [Full Text] [PDF]
|
|
|
|
 |
|
 S. Feldt, U. Maschke, R. Dechend, F. C. Luft, and D. N. Muller The Putative (Pro)renin Receptor Blocker HRP Fails to Prevent (Pro)renin Signaling J. Am. Soc. Nephrol., April 1, 2008; 19(4): 743 - 748. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Feldt, W. W. Batenburg, I. Mazak, U. Maschke, M. Wellner, H. Kvakan, R. Dechend, A. Fiebeler, C. Burckle, A. Contrepas, et al. Prorenin and Renin-Induced Extracellular Signal-Regulated Kinase 1/2 Activation in Monocytes Is Not Blocked by Aliskiren or the Handle-Region Peptide Hypertension, March 1, 2008; 51(3): 682 - 688. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. H. Gradman and R. Kad Renin inhibition in hypertension. J. Am. Coll. Cardiol., February 5, 2008; 51(5): 519 - 528. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Bader Spotlight on Renin: The second life of the (Pro)renin receptor Journal of Renin-Angiotensin-Aldosterone System, December 1, 2007; 8(4): 205 - 208. [PDF]
|
|
|
|
|
|
A. J. Danser Renin Academy in Focus Journal of Renin-Angiotensin-Aldosterone System, December 1, 2007; 8(4): 212 - 212. [PDF]
|
|
|
|
 |
|
 H. Kobori, M. Nangaku, L. G. Navar, and A. Nishiyama The Intrarenal Renin-Angiotensin System: From Physiology to the Pathobiology of Hypertension and Kidney Disease Pharmacol. Rev., September 1, 2007; 59(3): 251 - 287. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Moore, P. Dicker, J. K. O'Brien, M. Stojanovic, R. M. Conroy, A. Treumann, E. T. O'Brien, D. Fitzgerald, D. Shields, and A. V. Stanton Renin Gene Polymorphisms and Haplotypes, Blood Pressure, and Responses to Renin-Angiotensin System Inhibition Hypertension, August 1, 2007; 50(2): 340 - 347. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. H. Jan Danser, W. W. Batenburg, and J. H. M. van Esch Prorenin and the (pro)renin receptor--an update Nephrol. Dial. Transplant., May 1, 2007; 22(5): 1288 - 1292. [Full Text] [PDF]
|
|