Introduction THE INTRACELLULAR SIGNALING OF THE

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1 THE INTRACELLULAR SIGNALING OF THE PARADOXICAL STIMULATION OF RENIN GENE TRANSCRIPTION CAUSED BY ANGIOTENSIN II Dr. Terebessy Tamás Semmelweis University Department od Orthopedics Semmelweis University PhD School Budapest, Tutor: Prof. Dr. László Rosivall Consultant: Dr. István Mucsi Introduction The more than 100 year old history of the renin-angiotensin system (RAS) begins with Tigerstedt and Bergman, who substructed a substance having pressor effect from the kidney and named it renin. [1]. The RAS is one of the most important factor of the blood pressure- and the sodium balance regulation. Drugs controlling its function are commonly used antihypertensive medications. Findings of the last two decades have confirmed the local existance of the RAS elements in several types of tissues [2-5]. Investigating the detailed functioning of tissue reninangiotensin systems a growth factor like effect of AngII has been identified. The AngII activates MAP kinase signaling pathways through AT 1 receptors [6], facilitates tyrosin phosphorillation [7], and enhances the transcription activity of protooncogenes (eg. c- fos gene) [8]. The main product of the RAS the AngII supresses renin expression in the kidney juxtaglomerular cells forming a negative feedback loop. However, a few publications present direct or indirect prooves for the existance of a paradoxical positive feedback regulation in certain primariliy pathologic conditions when AngII stimulates renin expression. Lou et all. found decreased expression of renin mrna in the rat hypothalamus following ACE inhibitor treatment. The observed regulation is contrary to the well known stimulating effect of ACE inhibitors on renin mrna in the kidney. [9]. In newborn rat cardiac myocites both mechanical stress and AngII treatment directly increased production of RAS elements such as the expression of renin genes in in vitro modells. [10]. Proximal tubular renin expression was increased significantly in rats after subtotal nephrectomy. This increase was largely reduced by ACE inhibition, suggesting that enhanced renin expression was 2

2 caused by AngII. [11]. The above mentioned results raise the possibility of a positive feedback loop in the regulation of tissue RAS in certain conditions. Sigmund and co-workers reported that in a transgenic mice harbouring the human renin gene driven by a 900 bp long piece of the human renin promoter AngII in pressor dose induced an almost two fold increase of the renin mrna expression. These results confirmed for the first time the paradoxical upregulation of renin expression by AngII and raise a need to clarify the molecular details [12]. The growth factor like effects of AngII (cell growth, migration, extracellular matrix production, stimulation of other growth factors), are activated through early and late intracellular signal transduction pathways, including the MAP kinase cascades and tyrosin kinases. MAP kinases include elements of serine/threonin protein kinase superfamily. In our present laboratory work we analysed the signaling mechanisms of ERK and JNK cascades employed by AngII. The ERK cascade is known as a mediator of growth- and differentiating stimuli, primarily as the signal transduction pathway of growth factor- and cytokine receptors with endogenous tyrosin kinase activity [13]. The p21ras small G- protein is the first element of the phosphorillation cascade, which together with Raf-1 kinase (MAPKKK kinase) - activates the enzyme craf-1. The activated craf-1 phosphorillates the MEK enzyme on a serin residue. The MEK dual kinase phosphorillates the ERK enzyme on the threonin and tyrosin residues. [14]. Activation of PAK enzyme is considered to be important in the activation of the JNK cascade via Rho family mambers [15, 16]. The JNK activation by AngII in cardiac fibroblast was found to be protein kinase Pyk2 dependent by Murasawa et all. The authors hypothetised a Pyk2-Rac1-PAK activation mechanism [17]. Schmitz and co-workers suggest an Nck adapter protein signaling complex (Rac-Nck-PAK) to be the firt step of the JNK cascade. The phosphorillation cascade of JNK system is similar to the ERK cascade. Its first element is p21-kinase (PAK), which is followed by the MEKK1-SEK1 system and finally JNK activation. [18]. The activated ERK and JNK enzymes translocated to the nucleus and regulate transcription and cell cycle via phosphorillation of transcription factors (c-jun, ATF-2, Elk-1) [19]. Berk and Corson reported five significant tyrosin kinase signal transduction pathways related to AngII in vascular smooth muscle cells: Src kinases, Janus kinase family, Focal adhesion kinase, Ca dependent tyrosin kinase, Receptor tyrosin kinases. The authors suggest to investigate in future studies the physiologic diversity and tissue specificity of the various tyrosin kinase signaling pathways [20]. Besides the promt vasoconstructive response AngII has other long term biological effects such as cell hypertrophy, proliferation, extracellular matrix production. Well known long term effect of AngII is the activation of several protooncogenes, of which most frequently mentioned ones are c-fos, c-jun, c-myc, erg- 1 [21, 22]. In the present work we analysed the signaling mechanisms of c-fos promoter employed by AngII. The cellular effects of AngII are widely investigated. However, the intracellular signal transduction pathways contributing to these effects are yet partly clarified. The role of certain signaling systems are discussed form different, often contradictory points of view. We aimed to set up an experimental model system, in which the intracellular signaling mechanisms of the AngII induced gene transcription can be examined. We studied the intracellular signaling pathways of the stimulation of c-fos and renin promoter activity caused by AngII in an in vitro modell. 3 4

3 Aims In our present work we investigated the possible intracellular signal transduction pathways of AngII induced gene transcription. Hypotheses were: AngII activates the c-fos promoter MAP kinase cascades are activated by AngII the PI 3 kinase and PKC enzymes are involved in the regulation of c-fos promoter stimulation MAP kinase systems contribute to the regulation of enhanced c-fos promoter activity. In our experiments it was aimed to set up a modell system where the positive feedback mechanisms of the AngII induced renin gene transcription can be observed. Regarding the intracellular signal transduction of the paradoxically upregulated renin transcription by AngII we assumed that: AngII activates the proximal renin promoter ERK and JNK cascades are involved in the regulation of renin promoter activation PKC enzyme is involved in the regulation of the renin promoter stimulation activation of tyrozin kinases contribute to the intracellular regulation of the observed mechanisms Cells Chinese Hamster Ovarium (CHO) and porcine proximal tubular cell cultures (LLC-PK1) were used in our experiments. The CHO cells were stably expressing the rat AT 1A receptors, while the LLC-PK1 cells expressing the rabbit AT 1 receptors. The CHO/AT 1A cells were the kind gift of T.J. Thekkumkara [23], the LLC-PK1/AT 1 cells were from R.C. Harris [24]. Plasmids 5 6 C-fos promoter was used when detecting the AngII induced gene transcription in transient transfection experiments. Reporter plasmid containing the proximal renin promoter was used for observing the intracellular signaling pathways of the paradoxical renin activation induced by AngII. Second reporter plasmids used for internal standard were expression vectors encoding the β-galaktosidase enzyme (Clontech, Palo Alto, CA). Timidin kinase minimal promoter (TKβ) and virus promoters (Rous sarcomavirus - RSVβ and Cytomegalovirus - CMVβ) control the β-galaktosidase enzyme transcription in the second reporter plasmid. Some of the plasmids encoded the cdna of a dominant negative (DN) mutant version of signaling proteins. The DN mutant protein binds the substrate of the adequate signaling protein thus competitively inhibiting its function. DN protein encoding plasmids DNRas, DNRaf, DNMEK inactivating the elements of the ERK cascade and plasmids DNRac1 és DNJNK inactivating the elements of JNK cascade were used in our experiments [24-27]. Plasmids encoding proteins that directly inhibit ERK and JNK enzymes were applied to clarify the role of MAP kinase cascades. CL100 plasmid includes the MAP kináz phosphatase (MKP1) enzyme that inactivates ERK, while JIP plasmid encodes the JNK binding domain of the murine JNK interacting protein that inhibits JNK. The role of c-src tyrozin kinase was observed using the expression vector encoding the c-src-n-terminal kinase (Csk) that inactivates c-src.

4 Methods Plasmid preparation Escherischia Coli (DH5α) competent cells were transformed with the plasmids using the heat shock method. The clones included the plasmid were selected on LB bacterium substrate containing 100 mg/ml ampicillin. Preparated plasmids were detected with restriction digestion. Plasmids were extracted with phenolchloroform and finally pured by ultracentirfuging on cesiumklorid gradient. The tranzient transfection Transient transfection is a widely spread method of examining transcriptional alterations caused by different agonists and also of observing the intracellular regulation of these alterations [28]. The gene of interest is allocated (transfected) into the cell with the help of a riporter plasmid, therefore the changes of the transcriptional activity of the gene due to administration of agonists will be detectable. Manipulation of the known inracellular signalig pathways allow to study the regulation of transcription. Our cells (CHO/AT 1A ; LLC-PK1/AT 1 ) were transfected with the calcium phosphat precipitation method. The use of second reporter plasmids The efficiency of tranmsfection has a high standard deviation (SD) even within the same experimental setting, despite of the precise standardization of the experimental environment. The use of second reporter as an internal standard is a common method to normalize high SD. The essence of the method is that the expression of the cotransfected second reporter is constant and depends only on the ratio of transfected cells but not on the used agonist or antagonist. The activity of the reporter of interest can be normalized on the activity of the internal standard. Using this method the error originating from the differences of transfectional efficiency can be minimalized. [29]. Western blot After 4 hours of serum starvation cells were preincubated with an antagonist or its vehiculum for 15 minutes and afterwards treated with AngII or its vehiculum. Cells were then scraped into Tryton lysis buffer and boiled in Laemmli buffer. Samples containing equal amount of protein were electrophoretised on 8% SDSpolyacrilamid gel and transferred onto a nitrocellulose membrane. Following blocking and intensive washing blots were incubated with the primary antibody, then another intensive washing was applied. Finally blots were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody and visualised by enhanced chemiluminescense. Statistics All transfections were performed in duplicate and repeated at least three times. The results are shown as mean±sd and expressed as a ratio of the luciferase activity in the AngII treated groups compared to vehiculum treated groups. Student s T test or one-way ANOVA (where appropriate) was used to analyse the data. Results AngII stimulates the transcriptional activity of the c-fos promoter through the AT 1 receptor T 3 CHO/AT 1A cells were transiently transfected with a luciferase reporter construct containing the c-fos promoter and then treated with AngII. AngII dose dependently increased the transcriptional activity of the c-fos promoter (10-10 M: 1,15±0,13; 10-9 M: 1,5±0,16; 7 8

5 10-8 M: 2,28±0,12; 10-7 M: 2,71±0,13; 10-6 M: 3,08±0,27; 10-5 M: 3,03±0,11; p<0,05). Candesartan, a specific antagonist of the AT1 receptor, abolished this effect completely (1,43±0,13; p: ns;). AngII and serum stimulated the activity of the β- galactosidase reporter plasmids A second reporter plasmid containing the β-galactosidase gene driven by the human timidin kinase minimal promoter (TKβ) was cotransfected with the c-fos promoter to eliminate the error originating from the different efficiency of transfection. During the evaluation of our results a consequent increase of the β- galactosidase activity was observed in the cells treated with 10-7 M AngII compared to the activity measured in the cells treated with vehicle (2,57±0,04; p<0,01; figure 6/A.). Similar results could be observed when the cells were co-transfected with the second reporter plasmids expressing β-galactosidase driven by the promoter of the Rous sarcomavirus (RSVβ) or the citomegalovirus (CMVβ) (RSVβ: 1,74±0,26; CMVβ: 3,77±1,4; p<0,05; figure 6/A.). The specific AT 1 receptor blocker candesartan prevented the increase of the β-galactosidase activity on all three promoters (10-7 M AngII M Candesartan: TKβ: 1,4±0,22; RSVβ: 1,17±0,25; CMVβ 1,43±0,55; p:ns in all cases). Our results suggest that AngII stimulates the activity of the timidin kinase minimal promoter and the viral promoters via the AT 1 receptor. We repeated the above experiment using serum treatment instead of AngII. Serum stimulated the c-fos promoter and similary to AngII also increased the β-galactosidase activity of all three promoters (TKβ: 2,62+0,28; RSVβ: 4,15+0,82; CMVβ: 7,41+2,58; p<0,05). The role of the ERK cascade in the activation of the c- fos promoter An expression vector producing the specific ERK inhibitor MAP kinase phosphatase (CL-100) was cotransfected with the c-fos luciferase reporter construct, that impeded the activation of the c- fos promoter caused by AngII (AngII: 3,17±0,93 vs. AngII + CL- 100: 1,06±0,2; p<0,01). In the next experiment PD98059 a substance inhibiting the ERK activator MEK enzyme - was administered prior to AngII. PD98059 partially inhibited the stimulation of the c-fos promoter (AngII: 2,36±093 vs. AngII + PD98059: 1,44±0,32; p<0,05). Further experiments were planned to test the effect of inhibition of the Ras G-protein and the Raf enzyme by the dominant negative (DN) mutant versions of the enzymes. Inhibition of Ras and Raf completely abolished the stimulatory effect of AngII on the c-fos promoter (pcdna 3. 3,17±0,93 vs. DNRas: 1,16±0,3 vs. DNRaf 1,36±0,31; p<0,01). The p21rac1 G-protein takes part in the activation of the c-fos promoter The expression vector encoding the DNRac1 G-protein inhibited the stimulatory effect of AngII on the c-fos promoter (pcdna 3 : 3,17±0,93 vs. DNRac1: 1,18±0,31; p<0,01). AngII activates the proximal renin promoter via the AT1 receptor LLC-PK1/AT 1 cells were transiently transfected with the reporter plasmid encoding the human proximal renin promoter and the cell were treated with AngII. As expected, AngII increased the transcriptional activity of the proximal renin promoter in a dose dependent manner (10-10 M: 1,9±0,14; 10-9 M: 2,63±0,64; 10-8 M: 3,01±0,56 ;10-7 M: 3,49±1,25; 10-6 M: 3,17±0,64; p<0,05; figure 1). The maximal stimulation caused by 10-7 M AngII was completely 9 10

6 prevented by preincubating the cells with candesartan (10-7 M Candesartan: 1,52±0,21; 10-6 M Candesartan: 0,81±0,31; p:ns; figure 1.). AngII stimulates the activity of ERK and JNK enzymes Western blot experiments were performed to determine whether AngII activates the MAP kinase intracellular signaling pathways in LLC-PK1/AT 1 cells. In our experimental modell 10-7 M AngII treatment caused 16,2±3,3 fold stimulation on ERK and 3,84±1,1fold increase on JNK activity (p<0,01 in both cases; figure 2.). AT 1 receptor blocker candesartan (10-7 M) prevented these effects completely. JNK but not the ERK cascade may be involved in the activation of the renin promoter The finding that AngII activates the MAP kinase cascades raised the possibility that the MAP kinase cascades may play a role in the transcriptional activation of the renin promoter caused by AngII. In our transfection assays neither the MEK antagonist PD98059 (2,88±1,17 vs. 2,4±0,68; p:ns), nor the cotransfection of the dominant negative mutant versions of members of the ERK cascade influenced the AngII induced renin promoter activation (DN-Ras: 3,31±0,83; DN-Raf: 2,83±0,84; DN-MEK: 2,93±0,86; p:ns). On the other hand presence of both the JIP and the DNJNK proteins inhibited the effect of AngII on the renin promoter (2,75±0,69 vs. 1,8±0,34 és 1,6±0,23; p<0,01; figure 3.). In the following experiment DNRac1 protein was cotransfected with the renin-luciferase reporter construct into LLC-PK1/AT 1 cells. Unexpectedly DNRac1 did not influence the effect of AngII on the 582 base pair long renin promoter (2,75±0,69 vs. 3,21±0,53; p:ns; figure 3.). 5 4,5 4 3,5 3 2,5 2 1,5 1 0,5 0 AngII Relatív luciferáz aktivitás Cand M 10-9 M 10-8 M 10-7 M 10-6 M 10-7 M 10-7 M M 10-6 M Figure 1. The effect of AngII on the transcriptional activity of the proximal renin promoter The role of PKC Our cells were transfected with the 582 bp long renin-luciferase reporter vector and prior to AngII treatment the specific PKC blocker bisindolylmaleimed (Bis) preincubation was performed. The Bis treatment did not inhibited the stimulatory effect of AngII (2,88±1,17 vs. 3,83±2,14; p:ns;). Then the cells were preincubated with PMA for 24h to deplete all isoforms of PKC. Similary to Bis, depletion of the PKC isoforms did not inhibit the observed effect of AngII. Finally stimulatory dose of PMA resulted a non significant increase on the transcriptional activity of proximal renin promoter (1,24±0,45; p:ns). : p < 0,

7 p44 p42 Relatív aktivitás Veh AngII Veh AngII :p<0,01 Vehikulum 10-7 M AngII p-erk p-jnk Figure 2. The effect of AngII on the activity of ERK and JNK enzymes Inhibition of tyrosin kinases partially blocked the effect of AngII In our next experiment administration of the tyrosine kinase inhibitor genistein to the cells transfected with the renin promoter resulted an aproximately 50% decrease of the AngII stimuli (2,88±1,17 vs. 1,57±0,45; p<0.01). Next we examined the role of the c-src tyrosin kinase in the activation of the renin promoter. The c-src-n-terminal kinase (Csk) phosphorillates - therefore inhibits the activity of - the c-src enzyme. Cotransfection of the expression vector encoding the Csk enzyme resulted a 30% inhibition on the 582 bp long renin promoter activation caused by AngII (2,75±0,69 vs. 2,09±0,42; p<0,01; figure 3.) :p<0,01 Vehikulum p54 p M AngII Relatív luciferáz aktivitás 4 3,5 3 2,5 2 1,5 1 0,5 0 pcdna3 Csk DN-Rac DN-JNK JIP JIP + genistein Figure 3. The role of inhibition of c-src and the JNK cascade on the paradoxical stimulation of renin promoter by AngII Relation of tyrosin kinases and the JNK In the last set of our experiments we investigated whether tyrosine kinases involved in the observed effect of AngII were upstream of JNK activation. Genistein inhibited AngII induced phosphorillation of JNK in western blot experiments (3,84±1,12 vs. 1,31±0,56; p<0,01; figure 4.). Furthermore, preincubation with genistein of the tubular cells cotransfected with the JIP plasmid prior to AngII treatment resulted in a 35% decrease of the fold stimulation of the renin promoter (2,75±0,69 vs. 1,75±0,48; p<0,01; figure 3.). This result was not significantly different form the inhibitory effect of either genistein or JIP alone. : p < 0,

8 Discussion When analysing our transient transfection results AngII was found to consistently stimulate the ß-galactosidase activity of the internal standard plasmids. After normalization the enhanced transcription activity on c-fos promoter seemed to be abolished due to the AngII induced ß-galactosidase increase. In case the agonist influences the activity of both the promoter of interest and the second reporter, the standardization leads to a systematic error. In a contrary situation when the agonist has an opposing effect on the observed promoter and on the second reporter the standardization may enlarge the magnitude of the effect of the agonist. In this procedure a not significant effect could be magnified to reach statistical significance. A systematic deviation in the transfection efficiency leading to a difference in the luciferase activity of the two groups (ie. agonist traeted vs. vechiculi treated) is unlikely. It can be concluded that abandoning the use of a second reporter does not influence the essesment of the target promoter activity to such extent as the normalization with the second reporter activity - altered by the agonist - influences it. Bias originating from not applying standardization manoeuvre are negligible compared to the systematic error obtained when results are corrected with agonist influenced second reporter activity. Therefore we did not use second reporter plasmid in our transient transfection experiments investigating intracellular signaling mechanisms. In our experimental model it has been demonstrated that ERK is phosphorillated and c-fos promoter activity is enhanced due to AngII. The results of our transfection experiments performed to investigate the relation of ERK and c-fos promoter activation show that AngII activates ERK through the classic Ras-Raf-MEK singnaling cascade. It has also been proved that AngII induced activation of the promoter is dependent of ERK and the p21rac1 Ang II Genistein p-jnk Relatív aktivitás Vehikulum Vehikulum AngII + + Figure 4. Inhibition of tyrosin kinases block the activation of JNK caused by AngII small G-protein is involved in the process. Our findings suggest that the Rac protein also participates in the AngII induced ERK activation raising the possibility of a cross-activation within MAP kinase systems. Sigmund and co-workers observed that in transgenic mice harbouring the human renin gene driven by the 900 base pair long piece of the human renin promoter AngII in pressor dose caused a two fold increase in the mrna expression of the renin transgene. In our experiments we presented the paradoxical effect of AngII on the renin promoter in an in vitro modell. It has been confirmed that - Vehikulum + : p<0,01 Genistein AngII p54 p

9 AngII enhances the transciptional activity of the renin promoter in a dose dependent manner through AT1 receptor. This activation was dependent on tyrosin kinases and JNK. Our finding that paradoxical activation of renin promoter by AngII could be observed in both transgenic mice and in an in vitro model supports the hypothesis that promoter regions responsible for inhibition of renin expression caused by AngII are located upstream of -900 basepair. We demonstrated for the first time that besides the PKA/cAMP system JNK is also able to stimulate the transcriptional activity of the proximal renin promoter. Our results prove that AngII activates both ERK and JNK enzymes. Inhibition of elements of the ERK cascade did not influence the effect of AngII on the renin promoter suggesting that ERK cascade does not play a role in this process. On the other hand, inhibition of the JNK enzyme by cotransfection either the dominant negative mutant version of the enzyme or the expression vector encoding the JNK interacting protein (JIP) decreased the activation of the renin promoter by approximately 40%. The activated JNK further activates several tarnscription factors such as c-jun, ATF-2 and Elk-1 (transciption factor binding Ets domain) leading to the stimulation of gene transcription. There are several known transcription factor binding sites on the proximal renin promoter that should be mentioned in relation of JNK activation. The Ets domain and the transcription factor binding site (-36/-20) published by Tamura and co-workers potentially sensitive to JNK activation and may explain the activation of the renin promoter caused by JNK activation. The p21rac-1 small G-protein is considered to be one of the very first members of the JNK cascade and also known that tyrosin kinases may activate the JNK enzyme via Rac-1 and PAK. Our previous observations suggest that Rac-1 takes part in the intracellular signaling of AngII. In this experiment cotransfection of the dominant negative mutant version of Rac-1 did not influence the effect of AngII that implies a Rac-1 independent activation of JNK. The non selective tyrosin kinase inhibitor genistein decreased the effect of AngII on the renin promoter that suggests AngII dependent activation of protein tyrosin kinases or transactivation of receptor tyrosin kinases in our experimental setting. Cotransfection of the expression vector encoding the Src protein tyrosin kinase inhibitor c-src-n-terminal kinase also partially blocked the stimulation of the renin promoter. Our preliminary results raise the possible role of c-src enzyme in this process. In the last set of experiments genistein blocked the activation of JNK caused by AngII. Moreover, no further inhibitory effect was observed when expression vector encoding the JIP enzyme and genistein were used simultaneously. Our results indicate that tyrosin kinases and JNK activate the renin promoter in the same intracellular signaling pathway and tyrosine kinases lay upstream. References 1. Tigerstedt R, Bergman PG: Niere und Kreislauf. Scand Arch Physiol 1898, 8: Dzau VJ: Multiple pathways of angiotensin production in the blood vessel wall: evidence, possibilities and hypotheses. J Hypertens 1989, 7(12): Hollenberg NK, Fisher ND, Price DA: Pathways for angiotensin II generation in intact human tissue: evidence from comparative pharmacological interruption of the renin system. Hypertension 1998, 32(3): Navar LG, Rosivall L: Contribution of the renin-angiotensin system to the control of intrarenal hemodynamics. Kidney Int 1984, 25(6): Rosivall L, Rinder DF, Champion J, Khosla MC, Navar LG, Oparil S: Intrarenal angiotensin I conversion at normal and reduced renal blood flow in the dog. Am J Physiol 1983, 245(3):F Eguchi S, Dempsey PJ, Frank GD, Motley ED, Inagami T: Activation of MAPKs by angiotensin II in vascular smooth muscle cells. Metalloprotease-dependent EGF receptor activation is required for activation of ERK and p38 MAPK but not for JNK. J Biol Chem 2001, 276(11): Huckle WR, Prokop CA, Dy RC, Herman B, Earp S: Angiotensin II stimulates protein-tyrosine phosphorylation in a calcium-dependent manner. Mol Cell Biol 1990, 10(12):

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