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Am J Physiol Renal Physiol 288: F117-F124, 2005. First published September 14, 2004; doi:10.1152/ajprenal.00333.2003
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Enhancer-dependent inhibition of mouse renin transcription by inflammatory cytokines

Li Pan, Yanping Wang, Craig A. Jones, Sean T. Glenn, Heinz Baumann, and Kenneth W. Gross

Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, New York

Submitted 16 September 2003 ; accepted in final form 7 September 2004


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Inflammatory cytokines have been shown to inhibit renin gene expression in the kidney in vivo and the kidney tumor-derived As4.1 cell line. In this report, we show that cytokines oncostatin M (OSM), IL-6, and IL-1{beta} inhibit transcriptional activity associated with 4.1 kb of the mouse renin 5'-flanking sequence in As4.1 cells. The 242-bp enhancer (–2866 to –2625 bp) is sufficient to mediate the observed inhibitory effects. Sequences within the enhancer required for inhibition by each of these cytokines have been determined by deletional and mutational analysis. Results indicate that a 39-bp region (CEC) containing a cAMP-responsive element, an E-box, and a steroid receptor-binding site, previously identified as the most critical elements for enhancer activity, is sufficient for the inhibition induced by IL-1{beta}. However, mutation of each of the three component sites does not abolish the inhibition by IL-1{beta}, suggesting that the target(s) of cytokine action may not be the transcription factors binding directly to these sites. This CEC region is also critical, but not sufficient, for the inhibition mediated by OSM and IL-6. These data suggest that the direct target of the associated cytokines may be coactivators interacting with transcription factors binding at the enhancer. Finally, we show that OSM treatment caused a 17-fold increase in promoter activity when only 2,625 bp of the Ren-1c flanking sequence were tested, in which the enhancer is not present. Three regions including –2625 to –1217 bp, the HOX·PBX binding site at –60 bp, and –59 to +6 bp have been found to contribute to this induction.

As4.1; oncostatin M; IL-6; IL-1{beta}

RENIN, A COMPONENT OF THE renin-agiotension system (RAS), plays a major role in regulating systemic blood pressure and electrolyte balance. Expression of the renin gene is highly regulated both tissue specifically and developmentally (9, 30). In adults the principal source of active renin found in the circulation derives from juxtaglomerular (JG) cells of the kidney.

We recently showed that 4.1 kb of Ren-1c 5'-flanking sequences are sufficient to correctly specify Ren-1c expression spatially and temporally within embryonic, extraembryonic, and adult tissues in mice containing the Ren-1c-GFP transgene (10). To identify important transcription factor-binding sites within the 4.1-kb sequence, we employed a kidney tumor-derived As4.1 cell line, which was generated from transgenic mice containing the mouse Ren-2 5'-flanking sequence fused to SV40 T antigen (31). A HOX·PBX-binding element at –60 bp and an enhancer at –2.6 kb have been found to be critical for high-level expression of the mouse renin gene (19, 20). The enhancer contains at least 11 cis-elements (termed Ea to Ek), which are necessary for full activity (16, 17, 27). Among these, a cAMP-responsive element (CRE) (Ed), which binds CREB/CREM and an adjacent E-box (Ee), which binds USF1/USF2, are the most critical (16). Mutation of either sequence motif results in an almost complete loss of enhancer activity. In addition, binding sites for retinoic acid receptor (RAR)/retinoic X receptor (RXR)/Ear2 (Eb and Ec), nuclear factor I (Ef, Eh, Ej and Ek), Sp1/Sp3 (Eg), and an unidentified transcription factor (Ei) are located within the enhancer and contribute to enhancer activity (13, 17, 28). A nuclear protein-Y binding site (Ea) was also identified at the 3'-end of the enhancer and shown to negatively regulate enhancer activity (27, 29).

Recent findings suggest that renin expression is regulated by inflammatory cytokines. Treatment of As4.1 cells with LPS-induced cytokines such as OSM, IL-6, IL-1{beta}, and TNF-{alpha} sharply decreases renin mRNA levels (2, 21, 34). Suppression of renin expression by OSM was also demonstrated in vivo, with kidney renin mRNA levels substantially reduced within 48 h in mice injected with a mouse OSM-expressing adenovirus vector (2). OSM and IL-6 belong to the IL-6 family of cytokines, which also includes leukemia inhibitory factor (LIF), IL-11, cardiotrophin, and ciliary neurotrophic factor. Members of this family bind receptor complexes containing a specific ligand-binding subunit and a common signal transducing subunit, gp130 (6, 33). Through Janus protein tyrosine kinases (JAKs) and MAPK, the two major signaling pathways of IL-6 type cytokines, ERK and STATs are activated (5). IL-1{beta} and TNF-{alpha}, via binding of the IL-1 and TNF-{alpha} receptors, respectively, engage common signaling cascades, leading to the activation of NF-{kappa}B and stress MAPKs including p38 MAPK and JNK (1, 32).

Here, we report that the inhibition of renin gene expression in As4.1 cells by cytokines such as OSM, IL-6, and IL-1{beta} is mediated through the enhancer. Moreover, sequences within the enhancer that are required for the inhibitory effect of each cytokine were determined. Our results also suggest that OSM, IL-6, and IL-1{beta} inhibit mouse renin transcription via, at least in part, an ERK-dependent pathway. Finally, we show that a construct containing only 2,625 bp of the Ren-1c flanking sequence responds to OSM with a 17-fold increase in promoter activity. Three regions including –2625 to –1217 bp, the HOX·PBX binding site at –60 bp, and –59 to +6 bp have been found to contribute to this induction.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cytokines and chemicals. Mouse OSM and human IL-6 were produced in transfected COS cell culture (7). Mouse IL-1{beta} and TNF-{alpha} were purchased from Sigma and R&D Systems, respectively. PD-98059, SB-203580, and 1-pyrrolidinecarbodithioic acid (PDTC) were obtained from Calbiochem.

Plasmid construction. Plasmids 4100 and AdTA, a pGL2-basic-derived plasmid containing the adenovirus E1b TATA box, are described in Ref. 19. Plasmids 2866, 2625, 117enh, 117, Enh-TA, 2777/2625, 2738/2625, 2699/2625, 2684/2625, 2829/2663, 2829/2672, 2829/2684, 117CEC, 117CEmC, 117enh/me, and 117enh/mcre are described in Ref. 16. Plasmids 1217, 197, 70, and 59 are described in Ref. 18. Plasmid 117mPH is the same as 117 except that the HOX·PBX-binding site is mutated (TAATAAATCAA->TAccgAcTCAA). Plasmid 2866/2538-TA or Enh-NS-TA was constructed by inserting the PCR fragment containing the Ren-1c sequence from –2866 to –2538 or a 90-bp PCR fragment containing a partial NFI-X cDNA sequence into SacI/BglII-digested AdTA or BglII-digested Enh-TA, respectively.

Cell culture and transient transfections. As4.1 cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and transfected using FuGENE 6 (Roche Applied Science). For each transfection in a 100-mm culture dish, 4.4 µg DNA including 4 µg of reporter plasmid and 0.4 µg of plasmid containing Rous sarcoma virus promoter driving {beta}-galactosidase (RSV-{beta}gal) were mixed with 8.8 µl of FuGENE reagent. Five hours after transfection, cells in a 100-mm dish were subcultured into ten 35-mm wells in six-well plates. After 16 h, cells were treated in serum-free medium with 100 ng/ml mouse OSM, 100 ng/ml human IL-6, 10 ng/ml mouse IL-1{beta}, or 10 ng/ml mouse TNF-{alpha}. If the inhibitors of signaling pathways were added, cells had been treated with inhibitors in serum-free medium for an hour before cytokines were added. Cells were then harvested after 24-h cytokine treatment, and luciferase (Luc) activities were measured using the Luciferase Assay System (Promega). Luc activity is normalized with {beta}-gal activity, which is measured using Galacto-Light Plus chemiluminescent reporter assay (Tropix), to correct differences for transfection efficiency between plasmids. Protein concentration was used to normalize Luc of each sample (untreated or treated) for each plasmid transfected. All transfection results represent the average ± SE of at least three separate experiments. Luc activity for construct 4100 serves as a standard in all experiments and is defined as 100. The cytokine effect in Fig. 3C is expressed as a percentage of [(Luc activity from cells treated with cytokine/Luc activity from untreated cells) x 100] .



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  		Fig. 3. Identification of sequences within the Ren-1c enhancer that are necessary for the inhibitory effects of OSM, IL-6, and IL-1{beta} on Ren-1c transcription. A: schematic representation of constructs containing either 5'-truncated, 3'-truncated, or mutated Ren-1c enhancer fused to the 117-bp Ren-1c promoter. Boxes a, b, c, e, d, f, g, h, i, j, and k represent cis-regulatory elements Ea, Eb, Ec, Ee, Ed, Ef, Eg, Eh, Ei, Ej, and Ek, respectively, which bind NF-Y, RAR/RXR/Ear2, RAR/RXR/Ear2, USF1/USF2, CREB/CREM, NFI, Sp1/Sp3, NFI, unidentified transcription factor(s), NFI, and NFI, respectively. B: As4.1 cells were transfected with constructs shown in A and treated with various cytokines for 24 h. Luc activities were then assayed and normalized as described under MATERIALS AND METHODS. Luc activity is expressed relative to that of plasmid 4100 (arbitrarily set to 100). C: data in B are replotted as cytokine effect (%), which is expressed as a percentage of [(Luc activity from cells treated with cytokine/Luc activity from untreated cells) x 100]. *Statistically significant differences (P < 0.05) relative to the previous longer promoter construct among constructs containing 5'-truncations or 3'-truncations. {dagger}, ¶Statistically insignificant differences (P > 0.05) relative to constructs 117R1 and 117enh, respectively. §Statistically significant differences (P < 0.05) relative to construct 117CEC. The statistical analysis is performed using Student's t-test.

 
EMSA. The EMSAs were performed as described previously (16). PolydI-dC was used as a nonspecific DNA competitor in EMSAs with Ren-1c CRE as the labeled probe, whereas PolydG·polydC was used with Ren-1c E-box as the probe. The nucleotide sequences of the probes used are as follows: Ren-1c CRE, 5'-CCCAATGACATCACTAACCAC-3'; and Ren-1c E-box, 5'-TAACCACGCAGATGGTGACC-3' (16). Each EMSA was performed at least three times, and similar results were obtained.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
OSM, IL-6, IL-1{beta}, and TNF-{alpha} inhibit transcriptional activity of 4.1 kb but not 2.6 kb of Ren-1c 5'-flanking sequence. To examine effects of cytokines on renin promoter activity, DNA constructs 4100, 2625, and AdTA (Fig. 1A) were transfected into As4.1 cells and either left untreated or treated with OSM, IL-6, IL-1{beta}, or TNF-{alpha} for 24 h. These cells were then analyzed for relative Luc activities. OSM, IL-6, IL-1{beta}, and TNF-{alpha} decreased transcriptional activity of 4.1 kb of the Ren-1c 5'- flanking sequence by ~79, 66, 87, and 98%, respectively (Fig. 1B, left). However, TNF-{alpha} seems to have inhibitory effect on general transcription since it inhibited transcriptional activity of a minimal promoter containing only the adenovirus E1b TATA box by about 83%, whereas other cytokines had either no or minimal stimulatory effect on this minimal promoter (Fig. 1B, right). Thus the specific inhibitory effect of TNF-{alpha} on transcriptional activity of 4.1 kb of the Ren-1c 5'-flanking sequence is ~88% [specific inhibitory effect by TNF-{alpha} = (1 – effect of TNF-{alpha} on 4.1 kb of Ren-1c promoter/effect of TNF-{alpha} on minimal TATA promoter) x 100% = (1–2%/17%) x 100%], which is very close to the inhibitory effect of IL-1{beta}. Because TNF-{alpha} has an additional inhibitory effect on general transcription in As4.1 cells, which complicates interpretation of results from transfection assays, an investigation of the mechanism of inhibition of Ren-1c transcription by TNF-{alpha} was not pursued further in this study. When only 2,625 bp of the Ren-1c flanking sequence were tested for the cytokine effect, instead of inhibition, treatment with OSM caused a 17-fold increase in promoter activity, whereas an approximately threefold or twofold Ren-1c promoter-specific increase was observed for IL-6 and IL-1{beta}, respectively (Fig. 1B, middle). These results suggest that the region between –2.6 and –4.1 kb is necessary for the inhibitory effects of cytokines on Ren-1c transcription.



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  		Fig. 1. OSM, IL-6, and IL-1{beta} inhibit the transcriptional activity of 4.1 kb but not 2.6 kb of Ren-1c. A: schematic representation of plasmids 4100, 2625, and AdTA. The HOX·PBX-binding site is shown by a vertical line and indicated by proximal promoter element (PPE). The enhancer (–2866 to –2625) is shown by a thicker vertical line. B: As4.1 cells were transfected with constructs 4100, 2625, and AdTA and treated with various cytokines in serum-free medium for 24 h. Luciferase (Luc) activities were then assayed and normalized as described under MATERIALS AND METHODS. Luc activity is expressed relative to that of plasmid 4100 (arbitrarily set to 100). *Statistically significant differences (P < 0.05) relative to the samples prepared from untreated cells, measured by Student's t-test.

 
The enhancer is sufficient for the inhibitory effects of OSM, IL-6, and IL-1{beta} on Ren-1c transcription. To test the role of the enhancer in the inhibition of Ren-1c expression by OSM and other cytokines, the construct 117enh (Fig. 2A), containing the Ren-1c enhancer placed directly upstream of the Ren-1c promoter (–117 to +6 bp), was transfected into As4.1 cells and treated with different cytokines. OSM, IL-6, and IL-1{beta} were capable of inhibiting transcriptional activity of this construct comparable to that observed for construct 4100 (Figs. 1B and 2B). The Ren-1c promoter (–117 to +6 bp) is not necessary for gaining the inhibitory effects by these cytokines since construct 2866/2538-TA (containing Ren-1c sequence from –2866 to –2538 placed upstream of the adenovirus E1b TATA box) still retained the inhibitory effects of all three cytokines in roughly proportional amounts although the basal expression level is dramatically reduced (Fig. 2B). A positional effect of the enhancer relative to the promoter was noted. When the enhancer is placed immediately upstream of the TATA box (plasmid Enh-TA), the OSM effect is significantly reduced, whereas effects of other cytokines remained about the same. However, when a nonrenin sequence was inserted to separate the enhancer from the TATA box at a distance that mimics the separation seen in construct 117enh or 2866/2538-TA for construct Enh-TA (plasmid Enh-NS-TA), the OSM inhibitory effect was back to ~70%. These results indicate that the enhancer is sufficient for the inhibition of Ren-1c by inflammatory cytokines and that different mechanisms may be involved in reducing enhancer function by the cytokines.



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  		Fig. 2. Enhancer is sufficient for the inhibitory effects of OSM, IL-6, and IL-1{beta} on Ren-1c transcription. A: schematic representation of constructs 117enh containing the Ren-1c enhancer (–2866 to –2625) inserted immediately upstream of the Ren-1c proximal promoter from –117 to +6, 2866/2538-TA containing Ren-1c fragment (–2866 to –2538) inserted upstream of the adenovirus E1b TATA box, Enh-NS-TA containing a 90-bp nonrenin sequence (NS) inserted between the Ren-1c enhancer and the adenovirus E1b TATA box, and Enh-TA containing the Ren-1c enhancer inserted immediately upstream of the adenovirus E1b TATA box. B: As4.1 cells were transfected with constructs shown in A and treated with cytokines in serum-free medium for 24 h. Luc activities were then assayed and normalized as described under MATERIALS AND METHODS. Luc activity is expressed relative to that of plasmid 4100 (arbitrarily set to 100). *Statistically significant differences (P < 0.05) relative to the samples prepared from untreated cells, measured by Student's t-test.

 
Sequences within the enhancer required for the inhibitory effects of OSM, IL-6, and IL-1{beta} on Ren-1c transcription. To identify the sequences within the enhancer necessary for the cytokine effects in combination with a 117-bp promoter, a series of constructs containing deletions and mutations in cis-regulatory elements within the enhancer (–2866 to –2625) (Fig. 3A) was tested in As4.1 cells. Progressive deletions from the 5' of the enhancer gradually decreased not only basal promoter activity but the inhibitory effect of OSM as well (Fig. 3, B and C). Noting that the basal promoter-Luc construct 117 responded to OSM with a 6.75-fold increase in expression, thus the construct with a deletion of the sequence from –2866 to –2699 (plasmid 2699/2625) is still suppressed by OSM, despite the calculated value of 99% for the OSM effect (Fig. 3C). Further deletion of CRE (Ed) from plasmid 2699/2625 resulted in almost complete loss of enhancer activity and the OSM effect, yielding an activity close to the OSM effect observed for plasmid 117. Complementary results were obtained from 3' deletions. Deletion of Ea and Eb (plasmid 2829/2663) did not significantly change basal expression and the OSM effect. Deletion of the sequence from –2672 to –2625 (plasmid 2829/2672) resulted in reduced basal activity and the OSM inhibition. Further deletion of the E-box (Ee) (plasmid 2829/2684) greatly lowered basal activity and completely abolished the OSM inhibition of enhancer activity. Similar results were observed with IL-6 treatment. The subregion (CEC) containing the seemingly most effective elements Ed, Ee, and Ec when tested alone in plasmid 117CEC retained a significant response. Mutation of Ec from 117CEC did not significantly change the IL-6 effect but did partially abolish the OSM effect. Moreover, mutation of the CRE (Ed) or E-box (Ee) from 117CEC completely abolished not only transcriptional activity of the CEC region but the OSM or IL-6 inhibitory effect as well (data not shown). Plasmid 117enh/mcre or 117enh/me, which is the same as 117enh except that the CRE or E-box is mutated, respectively, was also tested in As4.1 cells for the OSM or IL-6 effect. Both constructs showed a similar OSM or IL-6 response. Mutation of either the CRE or E-box greatly reduced both enhancer activity and OSM or IL-6 inhibition. These results indicate that the transcription factor-binding sites which are important for basal enhancer activity are also important for OSM or IL-6 inhibition. We conclude that the CRE and E-box are not only the most critical elements within the enhancer regulating basal expression of the Ren-1c gene but are also the most critical elements regulating the inhibition of Ren-1c expression by OSM and IL-6.

Effects of IL-1{beta} on the 117enh-based deletion and mutation constructs were also tested in As4.1 cells (Fig. 3, B and C). Deletions from either end of the enhancer did not seem to significantly reduce the inhibition until either the CRE or E-box was deleted. The 39-bp fragment from –2702 to –2663 containing CRE, E-box, and Ec (plasmid 117CEC) is sufficient for the inhibition by IL-1{beta}. Mutation of either the CRE or E-box in 117CEC abolishes not only basal expression but the cytokine inhibition as well (data not shown), whereas mutation of Ec only slightly decreased the inhibition. However, the CRE or E-box is not absolutely required for the inhibition. Mutation of either the CRE (plasmid 117enh/mcre) or E-box (plasmid 117enh/me) in plasmid 117enh did not cause a significant loss of the inhibition by IL-1{beta}. These results are consistent with the suggestion that OSM, IL-6, and IL-1{beta} may moderate the actions of a coactivator which binds and coordinates multiple transcription factors on the enhancer.

Effects of OSM or IL-6 on As4.1 nuclear protein binding to Ren-1c CRE or E-box. Because mutation of the CRE or E-box in plasmid 117enh significantly reduces the inhibition induced by OSM or IL-6 (Fig. 3C), we tested whether treatment of OSM or IL-6 changed binding of As4.1 cell nuclear proteins to the CRE or E-box, which has previously been identified as binding site for transcription factor CREB/CREM or USF1/USF2, respectively (16). As4.1 cells were either left untreated or treated with OSM or IL-6 for 15 min, 1 h, 3 h, or 8 h in serum-free medium. Nuclear extracts were then harvested and tested for their abilities to bind the Ren-1c CRE or E-box in EMSAs. As shown in Fig. 4A, no changes in the nuclear protein binding to CRE were observed for OSM treatment. However, an apparent decrease in the intensity of the As4.1 cell nuclear protein/E-box complex was observed at 8 h of OSM treatment (Fig. 4B). The lower band observed in EMSAs using the probe E-box represents a nonspecific protein/DNA complex (16). It is possible that the decrease in the affinity of nuclear protein binding to the E-box after OSM treatment contributes to the inhibition of renin enhancer activity by OSM. Treatment of As4.1 cells with IL-6 did not result in detectable changes in CRE or E-box binding activities (data not shown).



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  		Fig. 4. Effects of OSM on binding of nuclear proteins prepared from As4.1 cells to Ren-1c CRE (Ed) or E-box (Ee). EMSAs were performed using Ren-1c CRE (A) or E-box (B) as the probe and As4.1 cell nuclear extracts that had been left untreated (labeled as 0 on the top) or treated with OSM for 15 min, 1, 3, and 8 h. Specific nuclear protein/DNA complexes, which were demonstrated previously (16), are labeled by arrows, whereas nonspecific protein/DNA complexes are indicated by NS.

 
Role of ERK-dependent pathway in the inhibition of Ren-1c expression by OSM, IL-6, or Il-1{beta}. To test which of the major signal transduction pathways mediates the inhibition of Ren-1c enhancer activity by cytokines, As4.1 cells were transfected with construct 117enh and treated with an inhibitor of ERK-dependent pathway (PD-98059) or of p38 MAPK pathway (SB203580) 1 h before treatment with OSM, IL-6, or IL-1{beta}. Results show that PD-98059 treatment increases basal expression of the construct by about twofold (Fig. 5A), as well as significantly reduces the inhibition by OSM, IL-6, and IL-1{beta} (Fig. 5B), indicating the involvement of an ERK-dependent pathway in basal expression and the inhibition of Ren-1c enhancer activity by OSM, IL-6 and IL-1{beta}. However, PD-98059 did not completely abolish the inhibition by any of the cytokines, suggesting that other signal transduction pathways may also be involved. Treatment with SB-203580 did not significantly change the inhibition by either OSM or IL-6 but decreased the inhibition by IL-1{beta}, suggesting that the p38 MAPK pathway may contribute to the inhibition of renin enhancer activity by IL-1{beta}. Because IL-1{beta} can use the NF-{kappa}B pathway to transduce the signals, the inhibitor of this pathway, PDTC, was also tested for its ability to relieve the inhibition of expression from construct 117enh by IL-1{beta}. PDTC did not significantly change basal expression and only minimally reduced the IL-1{beta} inhibition, indicating that the NF-{kappa}B pathway is not a major transduction pathway responsible for the inhibition of renin gene expression by IL-1{beta}.



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  		Fig. 5. Examination of signal pathways involved in the inhibition of Ren-1c expression by OSM, IL-6, and IL-1{beta}. A: As4.1 cells were transfected with construct 117enh and treated with the inhibitor of ERK-dependent pathway (PD-98059, 100 µM), p38 MAPK pathway (SB-203580, 20 µM), or NF{kappa}B pathway 1-pyrrolidinecarbodithioic acid (PDTC, 100 µM) 1 h before treatment with OSM, IL-6, or IL-1{beta}. Effect of PDTC on OSM or IL-6-induced inhibition of promoter activity was not determined (n.d.). Luc activity is expressed relative to that of plasmid 117enh (arbitrarily set to 100). B: data in A are replotted as cytokine effect (%), which is expressed as a percentage of [(Luc activity from cells treated with cytokine/Luc activity from untreated cells) x 100]. *Statistically significant increase (P < 0.05) over control treated with same cytokine, measured by Student's t-test.

 
Regions from –2625 to –1217 and –59 to +6 and the HOX·PBX binding site are responsible for the induction of transcriptional activity of 2,625-bp Ren-1c 5'-flanking sequence by OSM. As shown in Fig. 1, construct containing only 2,625 bp of the Ren-1c 5'-flanking sequence had a reduced basal expression but gained a 17-fold increase in transcriptional activity specifically by OSM. To identify regions within the Ren-1c promoter responsible for this increase, As4.1 cells were transfected with a series of 5' deletion mutants from –2625 to –59 (Fig. 6A) and either left untreated or treated with OSM. Deletions from –1217 to –197, –197 to –117, and –117 to –70 resulted in decreases in basal expression (Fig. 6B), whereas deletions from –2625 to –1217 and –70 to –59 caused losses of OSM induction (Fig. 6, B and C). Construct 59 containing only Ren-1c sequence from –59 to +6 still responded to OSM by about twofold. Furthermore, mutation of the HOX·PBX-binding site within the region from –70 to 59 in plasmid 117 (plasmid 117mPH) resulted in an about threefold decrease in OSM induction. These results demonstrate that regions from –2625 to –1217 and –59 to +6 and the HOX·PBX-binding site are responsible for induction of transcriptional activity of the 2,625-bp Ren-1c 5' flanking sequence by OSM.



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  		Fig. 6. Regions from –2625 to –1217 and –59 to +6 and the HOX·PBX-binding site are responsible for induction of transcriptional activity of 2,625-bp Ren-1c 5'-flanking sequence by OSM. A: schematic representation of plasmids 2625, 1217, 197, 117, 70, 59, and 117mPH. The HOX·PBX-binding site is shown by a vertical line and indicated by PPE (proximal promoter element). B: As4.1 cells were transfected with constructs shown in A and treated with OSM in serum-free medium for 24 h. Luc activities were then assayed and normalized as described under MATERIALS AND METHODS. Luc activity is expressed relative to that of plasmid 4100 (arbitrarily set to 100). C: data in B are replotted as fold-induction by OSM, which is expressed as Luc activity from cells treated with OSM/Luc activity from untreated cells. *,{dagger}Statistically significant differences (P < 0.05) relative to the next longer promoter construct and 117, respectively, measured by Student's t-test.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Previous results by Baumann et al. (2) showed that treatment of mice with LPS induces a renal inflammatory response that is accompanied by a reduction in renin mRNA levels in the kidney. Inflammatory cytokines induced by LPS usually include OSM, IL-6, LIF, IL-1{beta}, and TNF-{alpha} (4, 14, 38). All of these cytokines with the exception of LIF were capable of reducing renin mRNA in As4.1 cells. OSM yielded the most prominent suppression while lesser inhibitions were obtained with IL-6, IL-1{beta}, and TNF-{alpha}. The inhibitory effects of OSM, IL-1{beta}, and TNF-{alpha} were shown to require sequences within the 4.1 kb of the Ren-1c 5'-flanking region (2, 21, 35). In this study, we show that the 4.1-kb sequence can also mediate the inhibition of renin gene expression by IL-6. Furthermore, we show that the Ren-1c enhancer is sufficient to mediate the inhibitory effects of OSM, IL-6, and IL-1{beta}. Results from analysis of deletions and mutations within the enhancer suggest that the transcription factor-binding sites important for enhancer activity contribute to the overall inhibition by cytokines. The small region from –2702 to –2663 (CEC) containing a CRE (Ed), an E-box (Ee), and a retinoic acid receptor-binding site (Ec) is critical for both enhancer function and inhibition by cytokines. A significant fraction of the inhibitory effect of either OSM or IL-6 appears to be mediated through the CEC region, whereas for IL-1{beta} the CEC region is sufficient for the complete inhibition of enhancer activity. However, it does not seem that the individual transcription factor binding to the Ed, Ee, or Ec site is the sole target of IL-1{beta} action as mutation of the corresponding site from construct 117enh does not abolish the inhibitory effect of IL-1{beta}. An alternative interpretation compatible with the above observations is that the cytokine effects are mediated through coactivators which act in concert with multiple transcription factors binding to the enhancer complex.

Regulation of gene expression by cytokines through modulation of coactivator activities has been previously reported. A recent report shows that the coactivator p300 mediates the induction of androgen-independent transactivation of the androgen receptor by IL-6 (3). Moreover, Puigserver et al. (22) reported that cytokines including IL-1 and TNF-{alpha} affect energy expenditure though p38 MAPK-mediated activation of PPAR{gamma} coactivator-1. Finally, Zhu and Ting (40) reported that the target of INF-{gamma} in mediating suppression of the collagen {alpha}2(I) promoter is the class II transactivator (CIITA), which is a master regulator of major histocompatibility complex II, Ii, and DM genes. CIITA does not bind DNA but interacts with DNA-binding transcription factors RFX, CREB, and NF-Y (39).

In this paper, we have shown that OSM had opposite effects in regulating renin promoter activities depending on the presence of the enhancer. OSM inhibits enhancer activity and renin gene expression, whereas it induces promoter activity if the enhancer is not present. Three regions including –2625 to –1217, the HOX·PBX binding site at –60, and –59 to +6 have been found to contribute to this induction. IL-6 and OSM, in part, share the same signaling pathways and have similar cellular effects; however, there are also considerable differences (36). The identification of factors involved in OSM but not in IL-6 action may help us in understanding the difference in the signaling of these two cytokines. Also, in non-renin- producing cells, the renin enhancer is usually nonfunctional (16). It will be of interest to test whether treatment with OSM results in expression of renin in these cells.

Kurtz and co-workers (8) reported that although IL-1{beta} inhibits renin expression in As4.1 cells, it has no direct effect on renin mRNA abundance in isolated native JG cells. In a later report, they found that TNF-{alpha} was capable of inhibiting renin expression in both As4.1 and isolated native mouse JG cells (34). Because these two cytokines use different receptors but share similar signaling pathways, it is possible that the native JG cells do not express the IL-1 receptors as suggested by the authors (34). A recent report by the same group also suggests that the inhibition of Ren-1c by TNF-{alpha} is mediated by CRE (Ed) (35). They have shown that transcription factor NF-{kappa}B activated by TNF-{alpha} can form complex with proteins binding to CRE (Ed). However, we have shown that CRE (Ed) is not the only target for IL-1{beta}, which can also activate the NF-{kappa}B pathway, as mutation of CRE (Ed) in plasmid 117enh does not significantly affect the IL-1{beta} effect (Fig. 3C).

Inhibitors of different signal pathways were tested to try to identify the signaling mechanisms involved in the inhibition of mouse renin expression by cytokines. Results indicate that the ERK-dependent pathway is at least partially responsible for the inhibition by OSM, IL-6, and IL-1{beta}. This result is consistent with previously reported Western blot analyses showing that all these cytokines are capable of inducing the phosphorylated ERKs in As4.1 cells (2). Lower levels of ERKs induced by IL-6 compared with OSM may explain the smaller inhibition of renin enhancer activity by IL-6. However, the ERK pathway inhibitor PD-98059 did not completely inhibit the cytokine response, suggesting other pathways may also be involved. Alternatively, PD-98059 was not effective in inhibiting all MEK1 activity. Furthermore, treatment of As4.1 cells with OSM or IL-6 did not detectably induce the phosphorylation of JNK or p38 MAPK (Wang and Baumman, unpublished data), suggesting that the JNK-dependent pathway is not responsible for the inhibition of the mouse renin gene by OSM or IL-6.

As4.1, a kidney tumor cell line isolated from transgenic Ren-2-Tag mice, was selected for the study of transcriptional regulation of the mouse renin gene because it has many features characteristic of the renin-expressing JG cells in the kidney, including the presence of renin-containing dense granules, expression of high levels of renin mRNA, and secretion of active renin protein (11, 31). In addition, As4.1 cells respond to physiological stimuli such as cAMP (12, 16, 31), cytokines (2, 21, 34), mechanical strain (23, 25), and endothelin-1 (24) in a manner consistent with what would be expected in vivo. Kurtz and co-workers (15) have also reported that ANG II inhibits renin gene transcription in As4.1 cells. However, Ryan et al. (26) showed that ANG II (100 nM) has no effect on renin transcription in As4.1 cells. The discrepancy between these two reports is probably caused by the difference in sensitivities of experimental methods used to determine mRNA levels. Kurtz and co-workers (15) used ribonuclease protection assays to measure the amount of renin mRNA and found that ANG II (100 nM) only attenuated renin mRNA level by ~30% compared with the less sensitive Northern blot analysis used by Ryan et al. (26).

In conclusion, this study demonstrates that the 242-bp renin enhancer is sufficient to mediate the cytokine-induced suppression of renin gene expression. Furthermore, results from mutational analysis indicate that multiple transcription factor-binding sites within the enhancer are required for cytokine inhibition. A 39-bp CEC region containing a CRE, an E-box, and a retinoic acid receptor-binding site is critical not only for enhancer function and renin expression but for cytokine inhibition as well. We also show that the ERK signaling pathway is involved in the inhibition of renin gene expression by cytokines. Finally, three regions have been identified to contribute to the induction of transcriptional activity of the 2,625-bp Ren-1c 5'-flanking sequence by OSM.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by National Institutes of Health (NIH) Grants HL-48459 (to K. W. Gross) and CA-85580 (to H. Baumann) and funds from the Bruce Cuvelier Family. L. Pan was supported by a postdoctoral fellowship from NIH. This research used core facilities supported in part by Roswell Park Cancer Institute's National Cancer Institute-funded Cancer Center Support Grant CA-16056.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. W. Gross, Dept. of Molecular and Cellular Biology, Roswell Park Cancer Institute, Elm and Carlton St., Buffalo, NY 14263-0001 (E-mail: gross{at}acsu.buffalo.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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