TY - JOUR
AU - Mucsi,, István
AB - Abstract Background. Angiotensin II (Ang II) down-regulates renin expression in juxtaglomerular cells, however, recent experimental evidence obtained in transgenic mice suggested that Ang II may ‘paradoxically’ increase transcriptional activity of the proximal renin promoter. Methods. To dissect signalling mechanisms contributing to the up-regulation of the proximal renin promoter by Ang II, porcine proximal tubular cells stably expressing the rabbit AT1 receptor (LLC-PK/AT1) were transiently transfected with a luciferase reporter construct containing the 582 bp long piece of the human renin promoter. Activation of mitogen-activated protein kinases (MAPK) was detected by western blotting using phospho-MAPK-specific antibodies. Results. Ang II dose-dependently stimulated the transcriptional activity of the human renin promoter (10−7 M Ang II: 3.50±1.25-fold stimulation). In these cells Ang II activated both extracellular signal-regulated kinase (ERK) and the c-Jun-N-terminal kinase (JNK). Inhibition of the ERK cascade did not reduce the stimulation of the renin promoter by Ang II, however, two expression vectors designed to inhibit the JNK pathway, the dominant negative JNK and the Jun-kinase interacting peptide inhibited the fold stimulation induced by Ang II (2.75±0.69 vs 1.6±0.23 and 1.8±0.34, respectively; P<0.01). Furthermore, genistein, a tyrosine kinase inhibitor, blocked the effect of Ang II both on the JNK and the promoter activation. Conclusion. Ang II may have a stimulatory effect on the proximal renin promoter in proximal tubular cells and this effect is mediated by tyrosine kinases and the JNK cascade. angiotensin II, human renin promoter, mitogen-activated protein kinases, renal tubular cell, transcriptional regulation, tyrosine kinases Introduction Angiotensin II (Ang II) suppresses renin expression in the juxtaglomerular (JG) cells forming a negative feedback loop in the systemic RAS [1]. In vitro observations raised the possibility that, at least in experimental model systems, a positive feedback loop may be formed in the regulation of tissue RAS. In the rat hypothalamus, expression of renin mRNA was suppressed by enalapril [2]. In cardiac myocytes both mechanical stress and Ang II treatment directly increased renin expression in vitro [3]. Furthermore, proximal tubular renin expression was increased significantly in rats after subtotal nephrectomy and this increase was largely reduced by ACE inhibition [4]. These results, however, in most cases provide only an indirect indication and further evidence is needed to substantiate the hypothesis postulating a potential positive feedback loop. If the existence of such ‘paradoxical’ positive feedback was confirmed this might be one potential mechanism whereby Ang II would contribute to the pathogenesis of progressive tissue fibrosis. Recent studies by Keen and Sigmund reported that in transgenic mice harbouring the human renin gene driven by a 900 bp long piece of the human renin promoter, Ang II in pressor dose induced an almost 2-fold increase of the human renin mRNA expression independent of the change in blood pressure. These results confine the ‘paradoxical’ up-regulation of renin expression by Ang II to the proximal promoter of the renin gene. The authors also discussed whether the molecular details of this ‘paradoxical’ stimulatory effect of Ang II are unknown and need to be clarified [5]. Regulation of the human renin promoter has been studied in several systems. These studies have identified numerous potential transcription factor binding sites within the proximal promoter [6,7]. Konoshita et al. [7] have mapped six binding sites, including ones that are potential targets for mitogen-activated protein kinase (MAPK) cascades, within the first 500 bp of the promoter. Furthermore, Tamura et al. [8] have suggested that c-Jun may contribute to the activation of the mouse proximal renin promoter through a tentative binding site identified between −36 and −20 bp immediately 5′ of the transcription start site of the mouse Ren-IC gene. Renal tubular cells express minimal or no renin under normal conditions; however, they are able to regulate the level of renin mRNA under low salt or high salt intake, or they can increase renin expression during chronic tubulointerstitial injury [9]. It has been suggested that regulation of renin expression in renal tubular cells is different from that in the JG cells [10]. Ang II regulates gene transcription through several intracellular signal transduction pathways, including the protein kinase C (PKC) pathway as well as the different MAPK cascades: the extracellular signal-regulated kinase (ERK1/2) [11] and the c-Jun N-terminal kinase (JNK) [12]. ERK1/2 is classically activated via the Ras-Raf-MEK pathway, but the molecular details of the activation of the JNK by G-protein coupled receptors are less well understood. Tyrosine kinases [13], the small GTP-ase p21-Rac1 and the p21-activated kinase (PAK) [12] and recently the adaptor protein Nck have been suggested to be involved [12]. In the present work we analysed signalling mechanisms employed by Ang II that may contribute to the ‘paradoxical’ up-regulation of the transcriptional activity of the human proximal renin promoter in an in vitro experimental model system in renal tubular cells. In transient transfection experiments using porcine proximal tubular cells stably expressing the rabbit AT1 receptor (LLC-PK/AT1) we have found that Ang II stimulated the transcriptional activity of several renin promoter–luciferase constructs. Inhibition of PKC and the ERK cascade did not block the stimulatory effect of Ang II. However, blocking tyrosine kinases or the JNK cascade abolished stimulation of the renin promoter. From these data we conclude that under experimental conditions Ang II may activate the renin transcription in renal tubular cells through a tyrosine kinase and JNK dependent pathway. Materials and methods Materials Fetal bovine serum (FBS) and HEPES were purchased from Life Technologies GmbH (Karlsruhe, Germany), Dulbecco's modified eagle medium (DMEM), geneticin, Ang II, genistein, bisindolylmaleimide I and phorbol 12-myristate 13- acetate were from Sigma-Aldrich Co. (St Louis, MO, USA) and PD98059 was from Calbiochem (Darmstadt, Germany). Candesartan was a kind gift from Astra-Zeneca (Mölndal, Sweden). The p-ERK (E-4) antibody was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA) and phospho-SAPK/JNK (Thr183/Tyr 185) antibody was from New England Biolabs (Beverly, MA, USA). Anti-β-actin antibody was from Sigma-Aldrich Co. Plasmids The pGL3-582 reporter construct contained the −582 to +16 fragment of the human renin promoter subcloned into the pGL3-basic luciferase reporter plasmid (Promega, Madison, WI, USA). Three other reporter plasmids, containing the 145 (−145 to +16), 892 (−892 to +16) and 2824 (−2824 to +16) bp long piece of the promoter in the pGL3-basic plasmid, were also tested. These constructs were a kind gift from Dr F. Pinet (INSERM, Paris, France) [14]. The dominant negative (DN)-Ras, DN-Raf, DN-MEK and DN-Rac1 plasmids have been described previously in detail [11]. The JNK-binding domain (amino acids 127–281) of the murine JNK interacting protein-1 (JIP) was amplified by PCR with pfu from a murine brain cDNA library (Clontech, Palo Alto, CA, USA). The resulting PCR product, with a 5′ Kozak sequence and a 3′ stop codon added on to its ends, was inserted between the EcoRI and HindIII sites of the cytomegalovirus-driven expression vector, pcDNA3 (Invitrogen, San Diego, CA, USA). The DN-JNK plasmid was a kind gift from Dr P. Andreka and Dr N. H. Bishopric (University of Miami, Miami, FL, USA) [15]. Plasmid sequences were verified by dideoxy-sequencing using Sequenase 2.0 (USB, Cleveland, OH, USA). Cell culture and transfection LLC-PK1 cells stably transfected with the rabbit AT1 receptor (LLC-PK/AT1; a kind gift from Dr R. C. Harris, Vanderbilt University, Nashville, TN, USA) were grown in DMEM supplemented with 10% FBS, 100 µg/ml streptomycin, 100 IU/ml penicillin and 600 µg/ml geneticin. For transfection the cells were plated onto 60 mm dishes. At 50% of confluence, transfection was performed using the calcium phosphate precipitation method with 2.5 µg reporter construct, 7.5 µg interfering plasmid or pcDNA3, the empty expression vector. The total amount of DNA was adjusted to 12.5 µg DNA/dish using pcDNA3. Sixteen hours later the cells were washed three times with phosphate-buffered saline (PBS) and were incubated further in serum-free DMEM. After 6 h the cells were treated with 10−7 M Ang II or its vehiculum for 21 h. In some experiments pharmacological inhibitors or their solvents were applied for 45 min prior to administration of Ang II. Finally, the cells were washed three times with ice-cold PBS and scraped into 250 μl ice-cold 100 mM KH2PO4 (pH 7.8) containing 1 mM DTT. Cell lysate was prepared by the freeze–thaw method and the cellular debris was removed by microcentrifuging at 14 000 r.p.m. (5 min, 4°C). Luciferase activity was assayed from the supernatant as described previously [11]. Western blot analysis After 4 h serum deprivation, cells were pretreated with specific inhibitors or DMSO and were then treated with 10−7 M Ang II for an additional 15 min. Samples were then scraped into 250 μl Triton lysis buffer [30 mM HEPES (pH 7.4), 100 mM NaCl, 10 mM EDTA, 1 mM EGTA, 50 mM NaF, 1% Triton X-100, 1 mM PMSF, 20 µg/ml leupeptin, 20 µg/ml aprotinin, 5 mM benzamidine, 1 mM sodium orthovanadate) and were boiled in Laemmli sample buffer for 5 min. Equal amounts of protein (∼10 µg) were then subjected to electrophoresis on an 8% SDS–polyacrylamide gel and transferred to nitrocellulose membrane (Bio-Rad). Blots were blocked in 5% non-fat milk or 5% albumin in TBS containing 0.1% Tween 20, then washed and incubated with the primary antibody, followed by extensive washing. Finally, blots were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody and visualized by enhanced chemiluminescence (Amersham). Equal loading and transfer were confirmed by staining both the gel and the membrane with Coomassie blue and Ponceau stains, respectively. In addition, equal loading was demonstrated by stripping the membranes and reprobing with anti-β-actin antibody (Sigma). The blots shown are representative of at least three similar experiments and semi-quantitative analysis was performed using Scion Image 4.0.2 software. The results of the densitometry are shown as normalized ratios of the measured optical density of the p-JNK and β-actin blots. Statistical analysis All transfections were performed in duplicate and repeated at least four times. The result are shown as mean±SD and expressed as a ratio of the luciferase activity in the Ang II-treated groups compared to vehicle-treated groups (fold stimulation). Student's t-test or one-way ANOVA (where appropriate) was used to analyse the data. Results Ang II stimulates the transcriptional activity of the human renin promoter via the AT1 receptor As expected, Ang II stimulated the luciferase activity of the 582 bp long renin promoter-reporter construct (pGL3-582) in a dose-dependent manner in LLC-PK1/AT1 cells. The strongest activation was measured at 10−7 M Ang II (3.49±1.25, P<0.05). Candesartan, a specific AT1 receptor blocker, completely abolished this effect (Figure 1). Similar stimulation was seen with three different renin promoter–luciferase constructs: specifically, fold stimulation of the transcriptional activity of the constructs containing 145, 892 and 2824 bp long pieces of the renin promoter were 2.18±0.3, 3.59±1.15 and 3.29±1.67, respectively. These results confirm the specificity of the observed effect and also suggest that the regulatory sequences required for the ‘appropriate’ inhibitory effect of Ang II lay further upstream in the gene. Fig. 1. Open in new tabDownload slide Ang II stimulates transcriptional activity of the human renin promoter via the AT1 receptor. LLC-PK/AT1 cells were transfected with the pGL3-582 plasmid then stimulated with Ang II for 21 h. Ang II stimulated the luciferase activity of this promoter in a dose-dependent manner; the strongest activation was measured at 10−7 M Ang II. Some groups of cells were pretreated with 10−7 M and 10−6 M candesartan, a specific AT1 receptor inhibitor. Results (mean±SD) are expressed as fold stimulation by Ang II and the asterisks indicate that the relative luciferase activity of the reporter construct was significantly higher in the Ang II-treated cells (P<0.05; n = 8, ANOVA). Fig. 1. Open in new tabDownload slide Ang II stimulates transcriptional activity of the human renin promoter via the AT1 receptor. LLC-PK/AT1 cells were transfected with the pGL3-582 plasmid then stimulated with Ang II for 21 h. Ang II stimulated the luciferase activity of this promoter in a dose-dependent manner; the strongest activation was measured at 10−7 M Ang II. Some groups of cells were pretreated with 10−7 M and 10−6 M candesartan, a specific AT1 receptor inhibitor. Results (mean±SD) are expressed as fold stimulation by Ang II and the asterisks indicate that the relative luciferase activity of the reporter construct was significantly higher in the Ang II-treated cells (P<0.05; n = 8, ANOVA). JNK but not the ERK cascade may be involved in the activation of the renin promoter Ang II induces MAPK activation in different systems and both ERK and the JNK cascade were shown to contribute to the transcriptional effects of the peptide [11,12]. Therefore, we set out to study the induction of MAPK phosphorylation, i.e. activation in tubular cells by Ang II. LLC-PK/AT1 cells were stimulated with 10−7 M Ang II and western blots using phospho-MAPK specific primary antibodies were performed. Ang II induced a 16.2±3.3-fold increase in ERK phosphorylation and a 3.84±1.1-fold increase in JNK phosphorylation indicating the activation of both kinases (Figure 2). The stimulation of both ERK and JNK activity by Ang II was abolished by preincubation of cells with candesartan (data not shown) suggesting clearly that MAPK activation by Ang II is an AT1-mediated effect in our tubular cells. Fig. 2. Open in new tabDownload slide Ang II induces the phosphorylation of the extracellular signal-regulated- and the c-Jun-N-terminal kinases. LLC-PK/AT1 cells plated on 6 cm dishes were treated with 10−7 M Ang II or with vehiculum for 15 min. MAP kinase phosphorylation was assessed by western blotting using phospho-MAPK specific antibodies. The same membranes were reprobed for β-actin. Blots shown are representative of three similar experiments. Blots were scanned and semi-quantitative analysis of the western blots was performed using Scion Image 4.0.2 software. Optical density results are normalized to the amount of β-actin. Results (mean±SD) are expressed as fold stimulation by Ang II and the asterisks show that the phosphorylation of the enzymes were significantly higher in the Ang II-treated cells compared with controls (P<0.01; n = 8, Student's t-test). Fig. 2. Open in new tabDownload slide Ang II induces the phosphorylation of the extracellular signal-regulated- and the c-Jun-N-terminal kinases. LLC-PK/AT1 cells plated on 6 cm dishes were treated with 10−7 M Ang II or with vehiculum for 15 min. MAP kinase phosphorylation was assessed by western blotting using phospho-MAPK specific antibodies. The same membranes were reprobed for β-actin. Blots shown are representative of three similar experiments. Blots were scanned and semi-quantitative analysis of the western blots was performed using Scion Image 4.0.2 software. Optical density results are normalized to the amount of β-actin. Results (mean±SD) are expressed as fold stimulation by Ang II and the asterisks show that the phosphorylation of the enzymes were significantly higher in the Ang II-treated cells compared with controls (P<0.01; n = 8, Student's t-test). These findings raised the possibility that the MAPK cascades may play a role in the transcriptional activation of the renin promoter caused by Ang II. However, the MEK antagonist PD98059 at 6 × 10−5 M concentration, which inhibited ERK phosphorylation induced by Ang II, did not inhibit the effect of Ang II on the promoter (2.88±1.17 vs 2.4±0.68, NS; Figure 4). To confirm that the ERK cascade plays no role in this process, expression vectors encoding dominant-negative mutants of members of the ERK cascade (DN-Ras, DN-Raf and DN-MEK) were co-transfected with the pGL3-582 luciferase construct. Fold stimulation in the presence of DN-Ras, DN-Raf and DN-MEK (3.31±0.83, 2.83±0.84 and 2.93±0.86-fold, respectively) was not different from fold stimulation in cells co-transfected with pcDNA3, the empty expression vector, confirming that the ERK cascade plays no role in the activation of the proximal renin promoter by Ang II. Next we examined if the JNK cascade contributes to the transcriptional activation of the renin promoter by Ang II. Plasmids designed to block the JNK cascade, namely DN JNK or JIP, the JNK-binding domain of the murine JNK-interacting protein-1 were co-transfected with the pGL3-582 construct. JIP inhibits JNK, but not p38 kinase or ERK, by retaining JNK in the cytoplasm. Importantly, both DN JNK and JIP substantially inhibited the fold stimulation induced by Ang II (2.75±0.69 vs 1.6±0.23 and 1.8±0.34, respectively; P<0.01; Figure 3). Similar results were obtained by using the JNK inhibitor compound, SP600125 (data not shown). Fig. 3. Open in new tabDownload slide Effect of inhibition of the small GTP-binding protein p21-Rac1 and of c-Jun-N-terminal kinase on Ang II-induced renin promoter activation. LLC-PK/AT1 cells were co-transfected with the pGL3-582 construct and either the empty expression vector pcDNA3 or expression vectors coding for DN-Rac, DN-JNK and JIP. Results (mean±SD) are expressed as fold stimulation by Ang II in each group co-transfected with inhibitory plasmids compared to the empty expression vector. The asterisks indicate that the fold stimulation by Ang II was significantly lower than in the controls (P<0.05; n = 8, ANOVA). Fig. 3. Open in new tabDownload slide Effect of inhibition of the small GTP-binding protein p21-Rac1 and of c-Jun-N-terminal kinase on Ang II-induced renin promoter activation. LLC-PK/AT1 cells were co-transfected with the pGL3-582 construct and either the empty expression vector pcDNA3 or expression vectors coding for DN-Rac, DN-JNK and JIP. Results (mean±SD) are expressed as fold stimulation by Ang II in each group co-transfected with inhibitory plasmids compared to the empty expression vector. The asterisks indicate that the fold stimulation by Ang II was significantly lower than in the controls (P<0.05; n = 8, ANOVA). Fig. 4. Open in new tabDownload slide The effect of inhibition of PKC, tyrosine kinases and MEK on the Ang II-induced activation of the renin promoter (pGL3-582). LLC-PK/AT1 cells, transfected with the pGL3-582 plasmid, were pretreated with 5 × 10−7 M bis, 10−4 M genistein or with 6 × 10−5 M PD98059 then stimulated with 10−7 M Ang II for 21 h. Luciferase activity was assayed from the cell lysates. Results (mean±SD) are expressed as fold stimulation by Ang II in each groups. The bars represent the fold stimulation achieved by Ang II in cells pretreated with the different inhibitors. The asterisks indicate that the fold stimulation by Ang II in the cells pretreated with the specific inhibitors was significantly different from those in cells pretreated with the vehicle of the inhibitors (DMSO) (P<0.05; n = 12, Student's t-test). Fig. 4. Open in new tabDownload slide The effect of inhibition of PKC, tyrosine kinases and MEK on the Ang II-induced activation of the renin promoter (pGL3-582). LLC-PK/AT1 cells, transfected with the pGL3-582 plasmid, were pretreated with 5 × 10−7 M bis, 10−4 M genistein or with 6 × 10−5 M PD98059 then stimulated with 10−7 M Ang II for 21 h. Luciferase activity was assayed from the cell lysates. Results (mean±SD) are expressed as fold stimulation by Ang II in each groups. The bars represent the fold stimulation achieved by Ang II in cells pretreated with the different inhibitors. The asterisks indicate that the fold stimulation by Ang II in the cells pretreated with the specific inhibitors was significantly different from those in cells pretreated with the vehicle of the inhibitors (DMSO) (P<0.05; n = 12, Student's t-test). While in most systems p21Ras plays a role in ERK activation, another member of the p21GTP-ase family, p21Rac1, is thought to be involved in the activation of JNK [12,13]. To further elucidate the elements of the signalling cascade leading to activation of the renin promoter in our system N17Rac1, the DN mutant of p21-Rac1, was co-transfected. Interestingly, DN Rac1 had no influence on the effect of Ang II on the 582 bp renin promoter (2.75±0.69 vs 3.21±0.53, NS), suggesting that an alternative pathway may be responsible for JNK activation. Inhibition of PKC did not block the activation of renin transcription Ang II activates the PKC signalling pathway in several systems including tubular cells. To define the role of PKC in the stimulatory effect of Ang II, tubular cells transfected with the pGL3-582 promoter were preincubated with bisindolylmaleimide (Bis), a specific PKC inhibitor prior to Ang II stimulation. Bis (5 × 10−7 M) did not inhibit the stimulatory effect of Ang II on the human renin promoter. In fact, this intervention augmented the effect of Ang II by 30% (2.88±1.17 vs 3.82±2.14, NS; Figure 4). On the other hand, Bis, when used in the same concentration, prevented the stimulatory effect of phorbol 12-myristate 13-acetate (PMA) on the c-fos promoter in our tubular cells (data not shown), demonstrating that the inhibitor was able to block at least some PKC isoforms in our system. To further corroborate this finding, tubular cells were preincubated with 100 nM PMA for 24 h to deplete all isoforms of PKC. Similarly to Bis, depletion of the PKC isoforms did not inhibit the observed effect of Ang II on the pGL3-582 promoter (data not shown). To completely exclude the contribution of PKC to the observed effect of Ang II on the renin promoter we have performed a series of experiments using PMA to stimulate PKC in tubular cells. Stimulation of the tubular cells transiently transfected with our renin promoter–luciferase reporter construct with 10−7 M PMA caused no significant fold increase in the activity of the renin promoter (1.24 + 0.45, P = NS). These results together clearly exclude a role of PKC in mediating the stimulatory effect of Ang II on the proximal renin promoter. Inhibition of tyrosine kinases partially blocks the stimulatory effect of Ang II It has been demonstrated recently that transactivation of receptor tyrosine kinases or activation of cytoplasmic tyrosine kinases may play a role in transducing a signal from the AT1 receptor into the cell [16]. Consequently, we examined the effect of tyrosine kinases on the transcriptional regulation of the renin promoter by Ang II. Preincubation of tubular cells with 10−4 M genistein, a potent tyrosine kinase inhibitor, decreased the fold stimulation of Ang II by almost 50% (2.88±1.17 vs 1.57±0.45, P<0.01; Figure 4). Tyrosine kinases may lay upstream of JNK Several studies have recently reported that Ang II activates JNK via multiple tyrosine kinase pathways [12,13]. In the last set of experiments we investigated if tyrosine kinases involved in the observed effect of Ang II were upstream of JNK activation. Genistein inhibited the Ang II-induced phosphorylation of JNK in western blot experiments (3.84±1.12 vs 1.31±0.56, P<0.01; Figure 5). Furthermore, preincubation with genistein of the tubular cells co-transfected with the JIP plasmid prior to Ang II treatment resulted in a 35% decrease of the fold stimulation of the pGL3-582 promoter (2.75±0.69 vs 1.75±0.48, P = 0.006; Figure 3) that was not significantly different from the inhibitory effect of either genistein or JIP alone. These results altogether suggest that tyrosine kinases and JNK are arranged in a vertical, hierarchical signalling system where tyrosine kinases lay upstream of JNK activation. Fig. 5. Open in new tabDownload slide Genistein prevents Ang II-induced JNK phosphorylation. LLC-PK/AT1 cells plated on 6 cm dishes were preincubated with genistein or with DMSO (control). Cells were then treated with 10−7 M Ang II or with vehicle for 15 min. Western blots were performed with anti-phospho-JNK (New England Biolabs) antibody. The same membranes were reprobed for β-actin. Blots shown are representative of five similar experiments. Blots were scanned and semi-quantitative analysis of the western blots was performed using Scion Image 4.0.2 software. Optical density results are normalized to the amount of β-actin. Results (mean±SD; P<0.01; n = 8, Student's t-test) are expressed as fold stimulation by Ang II and the asterisks show that the phosphorylation of the enzyme was significantly different from the appropriate controls. The results are normalized for loading (β-actin abundance). Fig. 5. Open in new tabDownload slide Genistein prevents Ang II-induced JNK phosphorylation. LLC-PK/AT1 cells plated on 6 cm dishes were preincubated with genistein or with DMSO (control). Cells were then treated with 10−7 M Ang II or with vehicle for 15 min. Western blots were performed with anti-phospho-JNK (New England Biolabs) antibody. The same membranes were reprobed for β-actin. Blots shown are representative of five similar experiments. Blots were scanned and semi-quantitative analysis of the western blots was performed using Scion Image 4.0.2 software. Optical density results are normalized to the amount of β-actin. Results (mean±SD; P<0.01; n = 8, Student's t-test) are expressed as fold stimulation by Ang II and the asterisks show that the phosphorylation of the enzyme was significantly different from the appropriate controls. The results are normalized for loading (β-actin abundance). Discussion Recent results obtained in transgenic mice harbouring the human renin gene driven by a 900 bp long piece of the human renin promoter demonstrated that a pressor dose of Ang II caused a 2.1-fold increase in the human renin mRNA expression independent of the increase in blood pressure [5]. The results presented here, obtained in an in vitro transient transfection model employing a 582 bp long piece of the human renin promoter in proximal tubular cells, confirm and extend the above finding. We showed that in renal proximal tubular cells Ang II dose-dependently up-regulated the proximal renin promoter via the AT1 receptor employing a tyrosine kinase and JNK dependent pathway. The ‘paradox’ response evoked by Ang II on the proximal renin promoter seen in both transgenic mice and in our in vitro system suggests that the regulatory elements responsible for the down-regulation of the renin gene are missing from the first 900 bp of the promoter, but are present in genomic constructs containing very large amounts of flanking DNA [5]. In fact, several upstream regulator elements have been identified in both the mouse and the human renin gene [17,18]. It is therefore conceivable that some of these distant regulatory sequences, or a co-operation between distal and proximal regulatory sites mediate the physiologic inhibitory effect of Ang II in renin-secreting cells. In our in vitro model system, however, we showed for the first time that in addition to the protein-kinase A/cAMP system c-jun-N-terminal-kinase (JNK) also stimulates the transcriptional activity of the proximal human renin promoter. Further-more, we showed that in renal tubular cells JNK mediates the ‘paradoxical’ stimulatory effect of Ang II on the proximal renin promoter. Further studies in appropriate experimental settings need to be performed to test the hypothesis that the signalling mechanisms described in this work contribute to the ‘paradoxical’ up-regulation of the renin gene observed by others [2–5]. If this hypothesis is proven, that will add substantially to our understanding of the role of the local RAS in the pathogenesis of progressive tissue fibrosis. Recently, Muller et al. [19] reported that Ang II inhibited the renin promoter via PKC in the renin secreting As4.1 cells. They localized this effect within the first 2.9 kb of the promoter. Importantly, they showed that this inhibitory effect did not involve the AP1 binding sites localized to within the first 200 bp. On the other hand, we found that inhibition of PKC, either by using bisindolylmaleimide or by depleting the different PKC isoforms by a prolonged incubation of the cells with PMA, did not block Ang II-induced transcriptional activation. In fact, inhibition of PKC in our system modestly potentiated the effect of Ang II on the renin promoter. Unfortunately, Muller et al. have not used shorter promoter constructs to further narrow down the localization of the potential transcription factor binding site responsible for the observed effect. Consequently, it is difficult to explain the apparent differences between their results and ours. However, cell type differences may provide a possible explanation. Numerous studies, including our earlier work, reported that Ang II regulates gene transcription at least in part through different MAP kinases [11–13]. Data presented here confirm that in proximal tubular cells Ang II activates both ERK and JNK. Inhibition of the Ras-Raf-ERK cascade by multiple approaches failed to inhibit the effect of ANG, demonstrating that ERK is not involved in the regulation of the human renin promoter by Ang II. In contrast, inhibition of JNK either by overexpressing the JNK binding domain of the murine JNK-interacting protein (JIP) or a dominant inhibitory mutant of JNK1 prevented the effect of Ang II (Figure 4), clearly pointing to the role of JNK in the observed effect. JNK phosphorylates specific subsets of transcription factors, such as c-jun, ATF-2 [13] and also members of the Ets-domain transcription factor family, such as Elk-1 [20], resulting in transcriptional activation of target genes. Several potential transcription factor binding sites have been identified within the first 600 bp of the human renin promoter [7], including an Ets-domain binding site. Furthermore, Tamura et al. [8] have demonstrated a tentative binding site identified between −36 and −20 bp immediately 5′ of the transcription start site of the mouse Ren-IC gene. The presence of these potentially JNK responsive binding sites in the proximal renin promoter might explain downstream mechanisms linking JNK activation to enhanced promoter activity. These mechanisms, however, need further investigation. The small p21 GTP-ase Rac1 was previously described as an upstream mediator of JNK [12,13]. Moreover, tyrosine kinases were also shown to activate JNK via activating PAK through p21-Rac1 or directly [12]. We previously presented evidence that p21-Rac1 may participate in the intracellular signalling processes of Ang II [11]. However, overexpressing the dominant inhibitory mutant of p21-Rac1 did not inhibit the effect of Ang II on the renin promoter in proximal tubular cells, and this contradicted our hypothesis on the potential involvement of Rac1 in the observed effect. Genistein, a non-selective tyrosine kinase inhibitor, prevented the Ang II-induced renin promoter activity, suggesting that tyrosine kinases are involved in this process. Transactivation of receptor tyrosine kinases (e.g. EGF receptor and PDGF receptor), through both Src-dependent and Src-independent mechanisms or activation of cytoplasmic tyrosine kinases may be responsible for this effect. Our preliminary results obtained in transient transfection experiments using an expression vector for the c-Src-N-terminal kinase (Csk) points to the potential involvement of the Src family of cytoplasmic tyrosine kinases in the observed effect of Ang II (data not shown). Further work to dissect the molecular mechanisms leading to increased JNK activity in our tubular cells upon Ang II stimulation and to identify the specific tyrosine kinases involved is under way in our laboratory. Finally, genistein abolished JNK phosphorylation and no further inhibition of the effect of Ang II was seen when JIP and genistein were used simultaneously. Based on these results we believe that the tyrosine kinases involved lay upstream of JNK in this system. In summary, we have presented evidence for the first time that Ang II can stimulate transcriptional activity of the proximal human renin promoter in an in vitro model system through a tyrosine kinase and JNK dependent mechanism. Activation of JNK in this system is independent of p21-Rac1 and occurs through a tyrosine kinase pathway. Based on the data presented here and also some indirect evidence reported in the literature we propose that paradoxical up-regulation of the renin expression by Ang II might occur in certain tissues under specific conditions and that this pathological feedback mechanism may contribute to an amplification of the activity of the local RAS and to the progression of tissue fibrosis. Further work, however, is needed to substantiate this hypothesis with direct experimental evidence. I. M. is a Békésy Postdoctoral Fellow of the Hungarian Ministry of Education. Part of this work was presented at the ERA/EDTA Congresses in Nice in 2000 and in Vienna in 2001. We are indebted to Dr F. Pinet for providing us with the renin promoter constructs, Dr R. C. Harris for the LLC-PK/AT1 cells, and Dr P. Andreka and N. H. Bishopric for the DN-JNK plasmid. We thank Peter Morsing, PhD (Astra-Zeneca, Sweden) for providing candesartan. We appreciate the technical help obtained from the Department of Medical Microbiology of the Semmelweis University. The skilful assistance of Dr Kata Buzasi and Sarolta Adamko is gratefully acknowledged. This work was supported by grants from OTKA (T042651, T034409 and T029260), ETT 138/98, 232/2000, FKFP 0316 and the Hungarian Kidney Foundation. Conflict of interest statement. None declared. References 1 Bader M, Ganten D. Regulation of renin: new evidence from cultured cells and genetically modified mice. J Mol Med 2000 ; 78 : 130 –139 2 Lou YK, Liu DT, Whitworth JA, Morris BJ. Renin mRNA concentration in rat hypothalamus is decreased by enalapril. Clin Exp Pharmacol Physiol 1995 ; 22 : 493 –495 3 Malhotra R, Sadoshima J, Brosius FC, 3rd, Izumo S. Mechanical stretch and angiotensin II differentially upregulate the renin-angiotensin system in cardiac myocytes in vitro. 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Role of p38 and JNK mitogen-activated protein kinases in the activation of ternary complex factors. Mol Cell Biol 1997 ; 17 : 2360 –2371 Author notes 1Department of Pathophysiology, Faculty of Medicine, Semmelweis University, Budapest, 2Hungarian Academy of Sciences and Semmelweis University Nephrology Research Group, 3Department of Oral- and Maxillofacial Surgery, Faculty of Dentistry, Semmelweis University, Budapest and 41st Department of Medicine; Faculty of Medicine, Semmelweis University, Budapest, Hungary Nephrol Dial Transplant Vol. 19 No. 9 © ERA-EDTA 2004; all rights reserved
TI - Angiotensin II activates the human renin promoter in an in vitro model: the role of c-Jun-N-terminal kinase
JF - Nephrology Dialysis Transplantation
DO - 10.1093/ndt/gfh388
DA - 2004-09-01
UR - https://www.deepdyve.com/lp/oxford-university-press/angiotensin-ii-activates-the-human-renin-promoter-in-an-in-vitro-model-NoteygNBFK
SP - 2184
EP - 2191
VL - 19
IS - 9
DP - DeepDyve
ER -