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Endocrinology Vol. 147, No. 12 6011-6018
Copyright © 2006 by The Endocrine Society

Interleukin-1ß Attenuates Renin Gene Expression Via a Mitogen-Activated Protein Kinase Kinase-Extracellular Signal-Regulated Kinase and Signal Transducer and Activator of Transcription 3-Dependent Mechanism in As4.1 Cells

Xuebo Liu, Qi Shi and Curt D. Sigmund

Department of Internal Medicine (X.L., C.D.S.), and Department of Molecular Physiology & Biophysics (Q.S., C.D.S.), Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242

Address all correspondence and requests for reprints to: Curt D. Sigmund, Ph.D., Departments of Internal Medicine and Physiology & Biophysics, 3181B Medical Education and Biomedical Research Facility, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242. E-mail: curt-sigmund{at}uiowa.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The precise mechanism by which cytokines such as IL-1ß negatively modulate expression of the renin gene remains incomplete. IL-1ß can repress renin transcription under both baseline and retinoic acid-stimulated conditions in As4.1 cells, a renin-expressing cell line derived from the kidney. This repression does not require a negative regulatory element present in the renin enhancer but is optimal in the presence of the entire renin enhancer. Three tandem copies of the retinoic acid response element is sufficient to attenuate the retinoic acid-response by IL-1ß. The decrease in retinoic acid-induced renin promoter activity in response to IL-1ß was blocked with the general tyrosine kinase inhibitor Genistein. IL-1ß caused an increase in the phosphorylation of ERK, but not p38MAPK or c-Jun N-terminal kinase. PD98059, an Erk kinase inhibitor, significantly decreased IL-1ß-mediated phosphorylation of ERK1/2, and attenuated the repression of baseline renin transcription in response to IL-1ß. PD98059 partially reversed the IL-1ß effect on retinoic acid-mediated transcription. To further investigate this mechanism, we searched the downstream effectors of ERK1/2 pathway. Although there was no effect of IL-1ß on the phosphorylation of ELK, Janus kinase 2, or signal transducers and activators of transcription (STAT) 1, IL-1ß significantly increased tyrosine-phosphorylation of STAT3, an effect attenuated by PD98059. STAT3 overexpression significantly repressed transcription of the renin gene, whereas small interfering RNA-mediated knockdown of STAT3 increased renin at baseline and attenuated the IL-1ß response. We conclude that in As4.1 cells, IL-1ß down-regulates renin gene expression via a mechanism involving the Erk-STAT3 pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE RENIN-ANGIOTENSIN SYSTEM (RAS) is a central mediator of blood pressure regulation in mammals. Renin is the rate-limiting step in the production of the vasoactive component-angiotensin-II, and consequently is under tight transcriptional, posttranscriptional and posttranslational control. Two regions of the renin 5' flanking region have been reported to be critically involved in regulating expression of the gene. Sequences proximal to the promoter that include binding sites for the basal transcriptional machinery are also reported to regulate the cAMP response and may be necessary for developmental expression (1, 2, 3). A second important and highly conserved regulatory region is located approximately 2.6 kb upstream of the mouse renin gene, and 11 kb upstream of the human renin gene (4, 5, 6). Originally identified as an enhancer of renin transcription, it has now become clear through extensive mutagenesis analysis that this enhancer consists of a complex organization of binding sites for both stimulatory and inhibitory transcription factors (7, 8, 9, 10, 11).

Transcription factors reported to stimulate renin promoter activity through the enhancer include members of the steroid hormone family of nuclear receptors such as retinoic acid receptor (RAR{alpha}) and retinoid X receptor, members of the cAMP response element binding protein (CREB)/ATF family including CREB and cAMP responsive element modulator, the E-box binding factors upstream stimulatory factor (USF)-1 and USF-2, and nuclear factor I (NFI) (8, 9, 11). Negative regulatory factors include nuclear factor Y (NF-Y), vitamin D receptor (VD3R) and the orphan nuclear receptor Ear2 (7, 10, 12). NF-Y, which normally acts as a stimulatory transcription factor for many genes, acts as a negative regulator because its binding site overlaps and blocks accessibility of the steroid hormone receptor binding site (10). This mechanism for repression by NF-Y is shared with other genes (13). VD3R, a member of the same transcription factor subfamily as RAR{alpha}, along with Ear2 both repress enhancer activity, perhaps through a mechanism involving the RAR response element (RARE) (7, 12). Negative regulation of the renin promoter by TNF{alpha} through NF-{kappa}B has also been reported, and may act through a mechanism involving CREB1 and the cAMP response element (CRE) site in the enhancer (14, 15). That the enhancer sequence is under extensive negative control is consistent with the tight regulation of renin transcription in response to physiological cues. However, whereas we have identified some of the transcriptional players regulating the renin gene, our understanding of the transcriptional and signaling mechanisms linking physiological stimuli to changes in renin expression remains very incomplete.

Cytokines play a central role in the regulation of inflammatory reactions and host defense responses. The RAS has been reported to be a potential regulator of cytokine production. For example, angiotensin-II stimulates production of TNF{alpha} and IL-6, and both angiotensin converting enzyme inhibitors and angiotensin receptor blockers can abrogate the stimulation of cytokine production by endotoxin (16). IL-1ß is a potent cytokine that can induce expression of a number of other inflammatory cytokines and can produce tissue injury similar to that observed in septic shock (17). A side effect observed in IL-1ß clinical trials is hypotension suggesting potential cardiovascular implications (18). There has been substantial conflict in the literature regarding the effect of IL-1ß on renin secretion. Some reports suggest that IL-1ß stimulates renin release and increases plasma renin activity (19, 20), whereas other studies report opposite results (21). It is therefore unclear whether this is a specific and direct response to IL-1ß, or an indirect effect caused by IL-1ß-mediated hypotension, or IL-1ß-mediated induction of nitric oxide, which can also induce renin secretion (22, 23, 24, 25). However, it is largely undisputed that IL-1ß significantly down-regulates expression of the renin gene via a transcriptional mechanism (26, 27). Moreover, Gross and colleagues (28) have reported that this response is optimal in the presence of the CRE, E-Box, and the RARE present in the renin enhancer. In an effort to clarify the importance of renin gene regulation by IL-1ß, we investigated the transcriptional and intracellular signaling mechanisms underlying the IL-1ß response in As4.1 cells, a cell line derived from the kidney that endogenously expresses renin mRNA. We report that IL-1ß decreases renin expression via a mechanism involving ERK1/2 and signal transducers and activators of transcription 3 (STAT3) but not Janus kinase 2 (JAK2)-STAT3.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture, transient transfection, and luciferase assay
As4.1 cells (CRL2193; American Type Culture Collection, Manassas, VA) were maintained in DMEM (Invitrogen Life Technologies, Carlsbad, CA) supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 mg/ml). Cells (2 x 105/well) were plated in six-well culture dishes 24 h before transfection in DMEM containing 1.0% fetal bovine serum. Cells were transfected using Fugen-6 (Roche, Indianapolis, IN). IL-1ß (100 pg/ml) or vehicle was added to the media 24 h after transfection, and kinase inhibitors were added 1 h before IL-1ß. Forty to 48 h after transfection, the cells were harvested and lysed using the luciferase kit (Promega, Madison, WI). Luciferase activity was determined and normalized to total cellular protein in the lysate. In experiments examining the effects of PD98059 or STAT3, we performed a dual luciferase activity analysis using firefly and Renilla luciferase plasmids using the Promega dual luciferase kit. Because in preliminary experiments we noted that PD98059 markedly effected the level of Renilla luciferase, we redesigned the transfections to control for transfection efficiency. In brief, a master transfection was performed in 10-cm diameter plates, 6 h later, the transfected cells were split by trypsin and replated into six-well dishes. After overnight incubation, the cells were then treated as indicated with PD, IL-1ß, or RA and then luciferase was measured 24 h thereafter. Luc activity was normalized with total cellular protein. The pcDNA-STAT3 construct was the generous gift of Dr. James E. Darnell, Jr. (29).

Western blot
Whole cells were lysed in buffer [25 mM HEPES (pH 7.5),150 mM NaCl, 5 mM EDTA, 2 mM Na3VO4, 1% Triton X-100, 0.5% NP-40 with protease inhibitors]. For nuclear proteins, cells were resuspended in hypotonic buffer [10 mM HEPES (pH 7.8), 10 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, with protease inhibitors] on ice, for 20 min, detergent was added to 0.5%, nuclei were pelleted, then suspended in nuclear protein extract buffer [50 mM HEPES (pH 7.8), 300 mM NaCl, 50 mM KCl, 0.1 mM EDTA, 10% (vol/vol) glycerol with protease inhibitors] on ice for 30 min, and spun with the supernatant retained. Fifteen to 30 µg of total cell lysate or nuclear proteins were used for SDS-PAGE, and proteins were transferred to an Immobilon-P membrane (Millipore, Billerica, MA). The membrane was incubated with primary antibodies at room temperature for 1 h and washed with TBS-T [10 mM Tris-HCl (pH 7.6), 150 mM NaCl, and 0.1% Tween 20]. It was then incubated with antimouse or rat IgG conjugated with horseradish peroxidase (Amersham Biosciences, Piscataway, NJ) at room temperature for 1 h, and the immune complex was visualized with the ECL plus system (Amersham Biosciences).

RT-PCR
Total RNA was prepared from As4.1 cells treated by IL-1ß and/or PD98059 with the RNeasy mini kit (QIAGEN, Valencia, CA). The reverse transcription reaction was performed with an Invitrogen Superscript III, and real-time quantitative PCR was performed with a Bio-Rad Icycler and TaqMan chemistry. The cycling conditions comprised 2 min at 50 C, 10 min at 95 C, and 40 cycles at 95 C for 15 sec and 60 C for 60 sec. To correct for sample-to-sample variations, internal control genes, pcDNA-renin plasmid, were used to normalize the mRNA concentration.

Small interfering RNA (siRNA)-mediated suppression of STAT3
We designed two double-stranded siRNA oligoribonucleotides (Integrated DNA Technologies, Coralville, IA). The first was directed against mouse STAT3 (5'-AAC AUC UGC CUG GAC CGU CUG dTdT-3'; 3'-dTdTG UAG ACG GAC CUG GCA GAC-5'), whereas the second was a control siRNA that was ineffective against mouse STAT3 (5'-AAC CAC UUC AUG AGC AUC AUG dTdT, 3'-dTdTG GUG AAG UAC UCG UAG UAC-5'). siRNAs were transfected into As4.1 cells (10 nmol of siRNA per well of a six-well plate) using Lipofectamine-2000 (Invitrogen) following the manufacturer’s protocol. Cells were transfected for 24 h, IL-1ß (0.1 ng/ml) was added to the medium, then incubated for another 24 h. The cells were then harvested and lysed for Western blot analyses.

Statistical analysis
All results were compared by one-way ANOVA using the Bonferroni post hoc tests, or Student’s paired t test when paired samples were used using the SigmaStat software (Systat Software Inc., Point Richmond, CA). In a few cases where the ANOVA test indicated that the equal variance and normality tests failed (the data did not exhibit a normal distribution), a paired t test was used. Values are shown as means ± SEM. A value of P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Renin is regulated by a highly conserved complex element located 2.6 kb upstream of the mouse renin gene, and 11 kb upstream of the human renin gene (4, 6). Initially termed the renin enhancer, the element consists of the binding sites for at least 10 different transcription factors, including a RARE required for induction of renin promoter activity by retinoic acid (9) (Fig. 1AGo). We and others previously demonstrated that IL-1ß down-regulates activity of a transiently transfected renin promoter-luciferase construct consisting of 4.1 kb of the renin 5'-flanking region containing the enhancer (26, 28, 30). IL-1ß down-regulates the baseline activity of the m4.1k renin promoter to 60% of control, and the retinoic acid-induced stimulation of the renin promoter by 50% (Fig. 1BGo). IL-1ß-mediated repression does not require the adjacent NF-Y binding site (element-a, m4.1kµa), which overlaps the RARE and acts as a negative regulatory element (10) (Fig. 1CGo). This suggests the repression by IL-1ß occurs independently of NF-Y. Consistent with previous reports demonstrating the importance of the RARE, its mutagenesis in the 4.1-kb construct (elements b+c, m4.1µRARE) markedly diminished baseline, retinoid-stimulated, and IL-1ß-mediated repression of the renin promoter (Fig. 1DGo) (9). The effect of IL-1ß on baseline transcription of a construct containing three tandem copies of the RARE fused to a minimal renin promoter (3XRAREm117) was only reduced to 80% of control, whereas the effect on retinoic acid-induced expression was similar to the 4.1k construct (Fig. 1EGo). This suggests that other sequences either in the enhancer, proximal promoter, or elsewhere in the 4.1k construct may be required for optimal IL-1ß mediated repression (see Discussion). Both the core promoter lacking the 3XRARE (m117) or a construct where the three tandem copies of the RARE was mutated (3XµRAREm117) exhibited 100-fold lower baseline transcriptional activity (close to the background of the assay, data not shown).


Figure 1
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  		FIG. 1. IL-1ß attenuated renin promoter activity. A, Schematic representation of constructs employed in this study. 4.1kLuc consists of 4.1 kb of mouse renin 5' flanking region fused to luciferase as previously reported (9 ). 117Luc contains the 117-bp basal renin promoter and 3XRARE117luc contains three copies of the RARE fused upstream of the basal promoter. The cross-hatched bar represents the enhancer which is illustrated schematically in the inset. The letters correspond to transcription factor binding sites identified by mutagenesis: a, NF-Y; b+c, RARE; d, CRE; e, E-box. Schematic diagrams of the wild-type (WT), m4.1kµa (µa) and m4.1kµRARE (µRARE) are shown. B–E, Transcriptional activity of the renin promoter in the indicated constructs in response to vehicle (filled black), IL-1ß (cross-hatch), RA (filled gray), RA plus IL-1ß (wavy). Because the data did not exhibit a normal distribution, statistical comparisons were made between IL-1ß vs. control, RA vs. control (*, P < 0.05) or between IL-1ß + RA vs. RA ({dagger}, P < 0.05) by paired t test (n = 4 for all panels). The relative decrease in transcriptional activity in response to IL-1ß or IL-1ß + RA is indicated under each construct. RSV, Rous sarcoma virus; WT, wild type.

 
Tyrosine kinase, protein kinase A (PKA), protein kinase C (PKC), and phosphatidylinositol 3-kinase (PI3 kinase) inhibitors were next used to investigate the intracellular signaling pathway involved in the IL-1ß response (Fig. 2Go). When cells were pretreated for 1 h with 10 µM genistein, a tyrosine kinase inhibitor, IL-1ß-mediated attenuation of the retinoic acid response was abolished (cross-hatched bar, Fig. 2Go). H7, an inhibitor of PKA and PKC, modestly inhibited the action of IL-1ß on renin promoter activity, whereas other inhibitors of PKC and PI3 kinase had no effect. To examine whether specific tyrosine kinase families modulate the effect of IL-1ß, we tested the function of PP2, an inhibitor the Src family of tyrosine kinases, and AG490, an inhibitor of the JAK family of tyrosine kinases on renin gene transcription. Neither inhibitor caused obvious changes in IL-1ß-mediated inhibition of retinoid-induced renin promoter activity (data not shown); and IL-1ß did not induce phosphorylation of JAK2 (Fig. 3AGo). We therefore hypothesized that the dual-specificity kinase, MAPKs might be involved in IL-1ß-regulated renin gene expression. Consistent with this, IL-1ß treatment increased the level of phosphorylated ERK1/2, but not p38 or c-Jun N-terminal kinase (JNK) (Fig. 3BGo). PD98059, a specific MEK (ERK kinase) inhibitor, modestly diminished the level of phosphorylated ERK1/2 at baseline but significantly blocked its increase in response to IL-1ß (Fig. 3CGo).


Figure 2
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  		FIG. 2. Effect of kinase inhibitors. As4.1 cells were transfected with 4.1kLUC, 24 h later were pretreated for 1 h with the indicated inhibitor before IL-1ß and/or RA treatment. Luciferase (Luc) activity was measured 24 h later. G, Genistein; C, calphostin; S, staurosporine; B, biosindolyl; H7, (1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride); Ly, Ly294003; W, wortmannin. Statistical comparisons were made by one-way ANOVA using the Bonferroni post hoc test comparing first IL-1ß, RA and IL-1ß+RA vs. control (*, P < 0.05, NS is not significant), and then comparing the effect of the inhibitors or RA vs. IL-1ß+RA ({dagger}, P < 0.05), (n = 3). RSV, Rous sarcoma virus.

 

Figure 3
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  		FIG. 3. IL-1ß induces phosphorylation of Erk1/2. A and B, As4.1 cells were treated with 0.1 ng/ml IL-1ß for 24 h, whole-cell lysates were prepared and subjected to Western blot using antibodies for either nonphosphorylated or phosphorylated (P) forms of JAK2, p38, JNK, and ERK1/2 as indicated. Con, Untreated; IL-1, cells treated with IL-1ß. C, As4.1 cells were pretreated with PD98059 (PD) (20 µmol/liter) for 1 h before incubation with 0.1 ng/ml IL-1ß for 24 h.

 
We therefore hypothesized that IL-1ß may repress activity of the renin promoter via a MEK-ERK-dependent mechanism. Baseline activity of the renin promoter in transfected As4.1 cells was significantly increased after treatment with PD98059 suggesting a potential tonic effect of the MEK-ERK pathway on renin expression (Fig. 4AGo). Importantly, PD98059 blocked the decrease in renin promoter activity (Fig. 4AGo), and significantly attenuated the decrease in endogenous renin mRNA (Fig. 4BGo) and endogenous renin protein (Fig. 4CGo) mediated by IL-1ß. Whereas IL-1ß effectively blocked the induction caused by retinoic acid, this effect could only be partially reversed by PD98059, suggesting that both MEK-ERK dependent and independent mechanisms may be involved (Fig. 4DGo).


Figure 4
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  		FIG. 4. PD98059 (PD) inhibits IL-1ß-induced repression of the renin promoter. As4.1 cells were pretreated with PD98059 (20 µmol/liter) for 1 h before incubation with 0.1 ng/ml IL-1ß or RA (1 µmol/liter) for 24 h. A, As4.1 cells were transfected with 4.1kLuc reporter gene for 24 h and treated as indicated (n = 6). Because the data did not exhibit a normal distribution, statistical analysis was performed by paired t test. *, P < 0.05 vs. control; NS, not significant vs. control. Identical results were obtained using a log transformation of the data and ANOVA. B, Endogenous renin mRNA was measured by real-time RT-PCR (n = 3). *, P < 0.05 comparing IL-1ß+PD vs. IL-1ß alone by t test. C, Renin protein was measured by Western blot analysis. The renin (R), actin (A), and size markers are identified. D, As4.1 cells were transfected with 4.1kLuc reporter gene for 24 h and treated as indicated (n = 6). Statistical analysis was performed by one-way ANOVA with Bonferroni correction for multiple comparisons. *, P < 0.05 vs. control; NS, not significant vs. control; **, P < 0.05 vs. RA; NS2, not significant vs. RA. ¶, When the results were reanalyzed by paired t test, there was a trend toward significance (P = 0.059) comparing IL + R + PD vs. IL + RA.

 
To rule out a nonspecific effect of PD98059 on IL-1ß-mediated repression of renin promoter activity and renin protein abundance, we further investigated downstream effectors of the MEK-ERK pathway that directly link it to renin expression in As4.1 cells. IL-1ß significantly increased the level of phosphorylated STAT3 (Fig. 5AGo), but not ELK or STAT1 (data not shown). To verify these findings, we tested the effects of PD98059 and IL-1ß on the phosphorylation of STAT3 (Fig. 5BGo). IL-1ß significantly induced phosphorylation of tyrosine 705 (Y705) and modestly induced phosphorylation of serine 727 (S727) of STAT3. Pretreatment with PD98059 attenuated Y705 phosphorylation in a dose-dependent manner, and to a much greater degree than S727.


Figure 5
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  		FIG. 5. IL-1ß stimulates tyrosine phosphorylation of STAT3. A, As4.1 cells were treated with 0.1 ng/ml IL-1ß for 24 h, whole-cell lysates were prepared and subjected to Western blot using antibodies for either nonphosphorylated or phosphorylated forms of STAT3. Con, Untreated; IL-1, cells treated with IL-1ß. B, As4.1 cells were left untreated or treated with IL-1ß for 24 h after pretreatment with PD98059 (PD) either alone (20 µmol/liter) or in combination (10, 20, or 40 µmol/liter) with IL-1ß. Total proteins were subjected to Western blot using specific antibodies against phosphoY705 or phosphoS727 of STAT3. ß-Actin was included as an internal control.

 
We next overexpressed STAT3 in As4.1 cells to assess its effects on renin gene transcription and protein expression. Cotransfection of pcDNA-STAT3 with either the 4.1 kb or 3XRAREm117 renin promoter-luciferase reporter plasmids in As4.1 cells suppressed renin promoter activity compared with control (Fig. 6AGo). Consistent with the results shown above, the effect of STAT3 was greater on 4.1 kb than 3XRAREm117 (54% vs. 71% of control). STAT3 overexpression also caused a decrease in endogenous renin protein (Fig. 6BGo).


Figure 6
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  		FIG. 6. STAT3 inhibits renin promoter activity. A, As4.1 cells were cotransfected with pcDNA-STAT3 (or empty pcDNA3.1) and either 4.1kLuc (*, P = 0.018, n = 8) or 3XRAREm117Luc. (*, P = 0.008, n = 8) as analyzed by paired t test. Data are presented as percentage of untreated control (Con). B, As4.1 cells were transfected with pcDNA-STAT3 and endogenous renin protein was measured by Western blot (*, P = 0.018 vs. control by t test, n = 3). Data are presented as a percentage of untreated control. The inset shows a typical Western blot.

 
To confirm the role of STAT3 on renin gene expression, we used siRNA to specifically knock down the expression of STAT3 in As4.1 cells (Fig. 7Go). We first tested the efficacy of an siRNA directed against STAT3 on total STAT3 and PY-STAT3 (Fig. 7AGo). In independent experiments, siRNA routinely reduced total baseline STAT3 by 50–75%, and reduced PY-STAT3 by 75–90%. After IL-1ß, total STAT3 remained unchanged, whereas PY-STAT3 was significantly reduced by siRNA. To assess the effects on renin expression, renin protein and PY-STAT3 was measured after transfection of either siRNA or a nonspecific mutant siRNA (µRNA). Treatment of As4.1 cells with the nonspecific siRNA (µRNA) was ineffective at reducing PY-STAT3 (Fig. 7BGo). Inhibiting STAT3 modestly increased baseline renin and attenuated the repression caused by IL-1ß (Fig. 7CGo). There was no effect on renin induced by the nonspecific siRNA. These results establish that STAT3 acts as a negative regulator of renin expression in As4.1 cells and suggests that IL-1ß represses renin expression at least partially through its effects on ERK1/2 and STAT3.


Figure 7
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  		FIG. 7. Silencing of STAT3 induces renin. A, As4.1 cells were transfected with a STAT3 siRNA and 24 h later were either treated with vehicle or IL-1ß (0.1 ng/ml). After and additional 24 h. total STAT3 and PY705-STAT3 were measured. ß-Actin was used as an internal control. B, As4.1 cells were transfected with either a STAT3 siRNA or an ineffective mutant STAT3 siRNA (µRNA). Twenty- four hours later, cells were either treated with vehicle or IL-1ß (0.1 ng/ml). After and additional 24 h. Phospho-tyrosine-STAT3, Renin and ß-actin levels were analyzed by Western blot. C, Quantification of three (n = 3) independent Western blots is shown. Data are presented as percentage of untreated control. Statistical analysis was performed by one-way ANOVA with Bonferroni correction for multiple comparisons first comparing all samples vs. untreated control (*, P < 0.05; NS, not significant), and then comparing IL-1ß + siRNA or IL-1ß + µsiRNA vs. IL-1ß ({dagger}, P < 0.05; NS2, not significant).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Sepsis is the response to severe infections mediated through systemic release of proinflammatory cytokines. Severe hypotension can accompany septic shock or septic syndrome, and affected patients can exhibit severe cardiovascular complications and multiple organ failure (31). The cytokine IL-1ß is a potent proinflammatory and immunomodulatory agent involved in a variety of physiological and pathological processes including sepsis (32). Because IL-1ß appears to be a potent regulator of renin synthesis and release, it may play a role in septic shock-induced hypotension through its effects on the renin-angiotensin system (21, 26, 27). Herein, we investigated the molecular mechanism by which IL-1ß causes an attenuation of renin synthesis.

Protein kinase inhibitors of various classes were used to identify pathways activated by IL-1ß. Genistein, but not inhibitors of PKA, PKC, or PI3K, suppressed IL-1ß-mediated attenuation of renin gene transcription suggesting that tyrosine kinases are activated in response to IL-1ß in As4.1 cells. Because genistein is a nonselective tyrosine kinase inhibitor, we further evaluated the IL-1ß response using inhibitors of specific tyrosine kinase pathways. These studies revealed that the Src and JAK family of tyrosine kinases, which play central roles in a number of stress response pathways are not required to mediate the IL-1ß response, whereas an inhibitor of the MAPK pathway abolished the baseline transcriptional response to IL-1ß. It is now clear that IL-1ß can activate three types of MAPK, ERK, p38, and the stress-activated protein kinase-JNK (33, 34). In As4.1 cells, IL-1ß increased the phosphorylation of ERK1/2, and the decrease in baseline renin promoter activity caused by IL-1ß was blocked by the specific MEK inhibitor PD98059. ERK-MAPKs play an important role in cell growth and proliferation, and the stimulation of a number of G protein-coupled receptors results in their activation. For example, endothelin-1 induces the activation of ERK-MAPK in rat aortic vascular smooth muscle (35) and can attenuate cAMP-induced renin release from isolated juxtaglomerular cells (36). We nevertheless recognize that because the inhibition of retinoic acid-mediated induction of the renin promoter by IL-1ß was only partially reversed by the inhibitor suggests that other ERK-independent pathways may also be involved.

Typically, G protein-coupled receptors and tyrosine kinase receptors transmit activating signals to the Raf-MEK-ERK cascade through different isoforms of the small GTP-binding protein Ras (37). Activated ERKs can then phosphorylate many substrates located in the nucleus, membrane and cytoskeleton (38). Among the important nuclear substrates for ERKs are the transcription factors Elk-1, a component of the ternary complex that along with serum response factor assembles at the serum response element of a variety of genes (39, 40), and STATs (41). Many cytokines, hormones, and growth factors use STAT signaling pathways to regulate diverse biological responses including development, cell proliferation, differentiation, and survival (41). Although there was no change in phosphorylation of Elk-1, we showed that IL-1ß modestly induced phosphorylation on S727 on STAT3, but markedly induced phosphorylation of Y705, the latter could be attenuated by PD98059. STAT3 inhibited renin promoter activity and lowered endogenous renin, and knocking down STAT3 with an siRNA attenuated the IL-1ß response. This suggests the potential utilization of the nonclassical JAK-STAT pathway through which STAT3 is phosphorylated at both sites by MEK and ERK in As4.1 cells. The involvement of the MEK-ERK pathway in activation or repression of STAT3 transcriptional activity has been previously reported (42, 43). The differential regulation of STAT3 phosphorylation at Y705 and S727 is quite complicated and apparently exhibits cell-specific and ligand-specific effects. Indeed, in our study, the phosphorylation of Y705 was particularly robust and blocked by PD98059. In some cells, activation of MEK-ERK correlates with an increase in S727 phosphorylation and a decrease in Y705 phosphorylation (44), whereas in other cells, inhibition of MEK activity has no effect on phosphorylation of Y705 (45). Serine phosphorylation of STATs also has differential effects on tyrosine phosphorylation and DNA binding activity depending on the cell type and stimulus (46, 47, 48). We recognize of course that PD98059 did not completely block STAT3 phosphorylation suggesting the involvement of other kinases downstream of IL-1ß.

The inhibitory effect of STAT3 is not unique to renin expression. STAT3 in cooperation with c-Jun silences Fas receptor expression in advanced human melanoma (49), down-regulates the expression of cyclin D during liver development (50), and inhibits dihydrotestosterone-induced prostate-specific antigen expression in human prostate cancer LNCaP cells, an action attenuated by specific knock-down of STAT3 using siRNA (51). Previous studies by Gross and colleagues (30) examining the regulation of renin expression by oncostatin M suggests the requirement for STAT5 and negative regulatory sequences proximal to the renin promoter in cooperation with distal enhancer sequences. However, the regulation of renin by STAT5 appears complicated by the observation that dominant interfering mutations of STAT5 also reduced baseline activity of the renin promoter, which was further depressed by oncostatin M. The action of oncostatin M apparently does not require STAT3 as assessed by a dominant-negative mutant. Interestingly, IL-6 also down-regulated renin expression, and IL-6 is known to be a potent stimulator of STAT3 (29, 30).

Because there are no apparent STAT3 binding sites in the promoter of the renin gene, how then does STAT3 inhibit renin promoter activity? Our data demonstrate that loss of the RARE from the enhancer in the 4.1-kb promoter attenuates the IL-1ß response. Although three tandem copies of the RARE appears sufficient for IL-1ß to attenuate the retinoic acid response, it was insufficient on its own to attenuate baseline promoter activity. Pan et al. (28) reported that the CRE, E-box, and RARE in the renin enhancer was sufficient for IL-1ß-mediated inhibition, and was necessary but not sufficient for inhibition of renin promoter activity by oncostatin and IL-6 (28, 30). Therefore, the RARE may be required, but not sufficient on its own, for mediating the renin transcriptional response to cytokines. What remains unclear is the requirement for specific transcription factors (CREB, RAR, USF1/2) that bind these sites. It is possible that the transcriptional mechanism regulating renin expression by IL-1ß, IL-6, and oncostatin may be similar to TNF{alpha}, which was first reported to be mediated through noncanonical interactions between NF-{kappa}B and the CRE (14), and more recently to inhibit both CREB1 binding to the CRE and by decreasing transactivation of the p65 subunit of NF-{kappa}B (15). Indeed, STATs have also been reported to bind and recruit coregulators and to act as coregulators themselves. For example, STAT3 and STAT5 have been reported to interact with SMRT (silencing mediator for RAR and thyroid hormone receptor) and other corepressors for unliganded nuclear hormone receptors such as RAR (52). A link between STAT5, RAR{alpha}, and STAT3 has been reported in leukemia (53). STAT3 has also been reported to be an intermediate between estrogen receptor and PIAS3 (protein inhibitor of activated STAT3) (54). Taken all together, these data suggest that activation of STAT3 by IL-1ß may either act as, or recruit corepressor(s) for transcription factors bound at the renin enhancer/promoter. Furthermore, this suggests an additional level of complexity of renin enhancer action involving the interplay between the enhancer binding proteins CREB, USF-1/2, and RAR{alpha}/retinoid X receptor, which bind to closely linked sites, their coactivators such as p300 and CREB-binding protein, and corepressors such as STAT3, PIAS3, and SMRT.

A complex picture is beginning to emerge on the negative regulation of the renin gene via the enhancer. The enhancer was originally identified by virtue of its classical enhancer-like activity and its importance was hypothesized based on its conservation across species (4, 5, 6). However it is becoming clear that the enhancer is a complex regulatory element responding to both stimulatory and inhibitory cues. Negative regulation is mediated by the binding of NF-Y to a site that overlaps the RARE (10), by the binding VD3R or an orphan nuclear receptor called Ear2 to the RARE (7, 12, 55), by the interaction of NF-{kappa}B with CREB and the CRE element (14), cytokines such as oncostatin through STAT5 (30), and IL-1ß through STAT3. Exactly how these transcription factors respond to physiological cues that are known to down-regulate the renin gene in vivo remains to be elucidated.

In closing, it is important to recognize that the studies performed herein and reported many times elsewhere (14, 15, 28, 30) were performed in As4.1 cells. These cells were originally cultured from a renin-expressing kidney tumor isolated from a transgenic mouse expressing the T-antigen oncogene under the control of the renin promoter (56). Although these cells are still considered the best model of the renin-expressing juxtaglomerular cell, they are fully transformed (i.e. tumorigenic), which could result in alterations in signaling pathways. Kurtz and colleagues (27) reported that IL-1ß decreases renin expression in As4.1 cells but has little effect in isolated juxtaglomeular cells. We thus recognize that As4.1 cells may respond differently than isolated primary juxtaglomerular cells in culture, albeit the mechanism accounting for this difference remains unclear. Interestingly, IL-1ß increases plasma renin in vivo but suppresses renin expression in other cell types (21). IL-1ß also causes hypotension (23, 24, 25), and release of nitric oxide (24), which can also stimulate renin release (22), making it difficult to unravel the precise mechanism for increased renin release. Kurtz and colleagues (14, 15, 57) have used As4.1 cells as a faithful model to investigate the mechanisms controlling the negative regulation of renin expression by another potent proinflammatory cytokine (TNF{alpha}) and by calcium. Consequently, any alteration in the signaling pathway in As4.1 cells may be very selective. Hence, once the detailed molecular mechanism regulating renin expression by cytokines is established in these cells and the transcription factors and interactions among them are identified, experiments can be performed to assess whether a similar mechanism is operant in vivo.


   Footnotes
 
This work was supported by grants from the National Institutes of Health (HL48058, HL61446, and HL55006). We gratefully acknowledge the generous research support of the Roy J. Carver Trust.

Disclosures: X.L., Q.S., and C.D.S. have nothing to declare.

First Published Online September 7, 2006

Abbreviations: CRE, cAMP response element; CREB, cAMP response element binding protein; JAK2, Janus kinase 2; JNK, c-Jun N-terminal kinase; MEK, ERK kinase; NF-Y, nuclear factor Y; PI3 kinase, phosphatidylinositol 3-kinase; PKA, protein kinase A; PKC, protein kinase C; RAR, retinoic acid receptor; RARE, RAR response element; RAS, renin-angiotensin system; siRNA, small interfering RNA; STAT, signal transducers and activators of transcription; USF, upstream stimulatory factor; VD3R, vitamin D receptor.

Received February 1, 2006.

Accepted for publication August 28, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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