Identification of a novel region in the proximal promoter of the mouse renin gene critical for expression

【摘要】  An enhancer at -2.6 kb and a HOX·PBX-binding site at -60 bp have been demonstrated to be critical to expression of the mouse renin gene ( Ren-1 c ) in As4.1 cells. In this report, we show that a region (-197 to -70) immediately 5' to the HOX·PBX-binding site is also critical for Ren-1 c expression. Deletion of this region in a construct containing 4.1 kb of the Ren-1 c 5'-flanking sequence resulted in a 99% reduction in Ren-1 c promoter activity in As4.1 cells, suggesting the pivotal role for the region in the regulation of the mouse renin gene. Electrophoretic mobility shift and supershift assays have identified two nuclear factor I-binding sites and a Sp1/Sp3-binding site within the distal portion of the region (-197 to -103). Mutation of these three sites caused a 90% decrease in Ren-1 c promoter activity. Mutational analysis and electrophoretic mobility shift assays have also identified three additional transcription factor-binding sites within the region from -103 to -69, each of which contributes to high-level expression of Ren-1 c in As4.1 cells. Finally, we have shown that the Ren-1 c enhancer is the target for endothelin-1 (ET-1)-induced inhibition of Ren-1 c expression and the transcription factor-binding sites in the proximal promoter are required for the maximal ET-1 inhibitory effect.

As4.1 cells; transcription factor; endothelin

【关键词】  Identification proximal promoter critical expression

RENIN, A COMPONENT OF THE renin-angiotensin system (RAS), plays an important role in regulating blood pressure and electrolyte homeostasis. Transcription of renin genes is tissue specifically and developmentally regulated ( 12, 32 ). Although mechanisms involved in this regulation are not well understood, the development of a kidney tumor-derived As4.1 cell line has facilitated the identification of important regulatory regions in the 5'-flanking regions of renin genes. This cell line was isolated from transgenic mice harboring a mouse renin ( Ren-2 ) 5'-flanking sequence fused to SV40-T antigen and has many features characteristic of the renin-expressing juxtaglomerular (JG) cells in the kidney, including expression of high levels of renin mRNA and secretion of active renin protein ( 13, 33 ).

A HOX·PBX-binding site at -60 bp has been found to be critical for Ren-1 c expression ( 21, 22 ). HOX9/10 members and PBX1b bind to the site in As4.1 cells ( 21 ). Another homeodomain protein, PREP1, forms a ternary complex with HOX·PBX by interacting with PBX directly. Furthermore, an enhancer located at -2.6 kb is also necessary for high-level expression of the Ren-1 c gene ( 22 ). This enhancer is conserved among humans, rats, and mice, although species-specific differences exist in the cis -acting regulatory elements within the enhancers (28, 39; Pan L, Liang P, and Gross KW, unpublished observations). Eleven transcription factor-binding sites have been identified within the 242-bp mouse enhancer. Among these, a cAMP-responsive element (CRE) and an adjacent E-box, which bind CREB/CREM and USF1/USF2, respectively, are the most critical elements for enhancer activity and Ren-1 c expression ( 19 ). Two TGACCT motifs, separated by 10 bp, are located downstream of the E-box ( 28 ). They have been shown to bind the retinoic acid receptor/retinoic X receptor and are important for both basal and retinoic acid-induced expression of the mouse renin gene ( 29 ). The nuclear Orphan receptor Ear2 is also capable of binding to these sites to negatively regulate renin gene transcription ( 17 ). A recent report has demonstrated that renin expression could also be downregulated by vitamin D treatment and implicates the TGACCT motifs as potential binding sites for vitamin D receptors ( 16, 31 ). Moreover, a NF-Y site has been located close to the 3'-end of the enhancer, which overlaps with the downstream TGACCT site ( 28 ). It has been shown that binding of NF-Y prevents the binding of transcription factors to the TGACCT site and thus inhibits enhancer activity ( 30 ). In addition, deletion of the distal portion of the mouse enhancer (-2866 to -2699) resulted in a 10-fold loss of enhancer activity ( 20 ). Four nuclear factor I (NFI)-binding sites, a Sp1/Sp3 site, and an unknown transcription factor-binding site have been identified within the region, each of which contributes to overall enhancer activity and Ren-1 c expression.

In addition to the enhancer and the HOX·PBX-binding site, a cAMP-responsive element-negative regulatory element (CNRE) has been identified at around -600 bp of the mouse renin genes ( 4, 18 ). The transcription factor LXR has been shown to bind to the CNRE and confer cAMP responsiveness ( 35 ). It is also suggested that CNRE is involved in gene-specific expression of mouse renin in the submandibular gland ( 11, 38 ). However, results from analysis of sequences and expression of natural renin gene variants found in the closely related mouse species Mus hortulanus argue against this ( 1, 2 ).

In this report, we identify a novel region (-197 to -70) in the proximal promoter of the Ren-1 c gene critical for its expression in As4.1 cells. Deletion of this region causes a 99% decrease in Ren-1 c promoter activity. Two NFI-binding sites and a Sp1/Sp3-binding site were identified in a more distal portion of the region (-197 to -103), whereas three transcription factor-binding sites including a CCAC motif and a CCTG motif were identified more proximally (-103 to -69). Mutation of the CCAC or CCTG motif causes an 85 or 78% decrease in Ren-1 c promoter activity, respectively, suggesting the importance of these two elements. These identified cis -acting elements in adjunction with the HOX·PBX site at -60 bp play a pivotal role in regulating the mouse renin gene, because mutations of all these sites result in a Ren-1 c promoter with only 0.24% of wild-type promoter activity. Finally, we have shown that the Ren-1 c enhancer is the target for endothelin-1 (ET-1)-induced inhibition of Ren-1 c expression, and the transcription factor-binding sites in the proximal promoter are required for the maximal ET-1 inhibitory effect.

MATERIALS AND METHODS

Plasmid construction. Plasmids 4100, 2625, Enh-TA, and TA were described previously ( 19, 21 ). Plasmids 4100 500/400, 4100 500/300, 4100 500/197, 4100 500/157, 4100 500/117, and 4100 500/70 were constructed by inserting the Sac I/ Hin dIII-digested PCR fragments containing the Ren-1 c sequences from -400 to +6, -300 to +6, -197 to +6, -157 to +6, -117 to +6, and -70 to +6, respectively, into the Sac I/ Hin dIII-digested m4.1kP-luc ( 28 ), a gift from Dr. C. D. Sigmund (University of Iowa College of Medicine). Plasmids 4100 197/94 and 4100 197/70 were constructed by inserting the Sac I-digested PCR fragments containing the Ren-1 c sequence from -500 to -94 and -500 to -70, respectively, into the Sac I-digested 4100 500/70. Plasmids 1217 and 59 were constructed by digesting 4100 and 4100mPa Hin dIII, ( 22 ), respectively, with Kpn I and then religating. Plasmids 197, 94, and 70 were constructed by inserting Sac I-digested PCR fragments containing the Ren-1 c sequence from -197 to +6, -94 to +6, and -70 to +6 into the Sac -I/ Hin dIII-digested pGL2-basic (Promega). All the site-specific mutants except 4100mPa were constructed by inserting the PCR fragments containing the mutations into the Sac I/ Hin dIII-digested m4.1kP-luc.

Cell culture and transient transfections. As4.1 and JEG-3 cells were grown in DMEM containing 10% fetal bovine serum and transfected with luciferase (Luc) reporter plasmid and plasmid containing Rous sarcoma virus promoter driving -galactosidase (RSV- gal) using FuGENE 6 (Roche) as described ( 19 ). Luc activity is normalized with -galactosidase activity to correct for differences in transfection efficiency between experiments. Transcriptional activities of plasmid 2625 in As4.1 and JEG-3 cells were compared relative to -galactosidase activity. For transfections involving ET-1 treatment, cells were either left untreated or treated with 10 nM ET-1 (Sigma) in serum-free medium 16 h after transfection. Cells were then harvested after 24-h ET-1 treatment. Results from all transfection assays are expressed as the means ± SE of at least three separate experiments.

EMSA. The EMSAs were performed as described previously ( 19 ). Poly(dI-dC) was used as a nonspecific DNA competitor in EMSAs with pRNFIU, pRCACC, or pRNFID as the labeled probe, whereas polydG·polydC was used in EMSAs with Pd or GC as a probe. Antibodies against Sp1 and Sp3 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); the NFI antiserum ( 27 ), generated against a bacterial fusion protein containing human CTF2 (an isoform of NFI-C), was kindly provided by Dr. N. Tanese (New York University Medical Center).

RESULTS

Region from -197 to -70 in the Ren-1 c promoter is essential for high-level expression. To identify regions in the 5'-flanking sequence important for Ren-1 c expression, a series of deletions were made on plasmid 4100, which contains 4.1 kb of the Ren-1 c 5'-flanking sequence upstream of a Luc reporter gene, and tested in As4.1 cells for their abilities to drive expression of Luc. Deletion of the sequence 5' to the enhancer (-4100 to -2866) resulted in a 50% decrease in Ren-1 c promoter activity, whereas small internal deletions covering the region from -2625 to -500 didn't significantly change expression levels (data not shown). However, deletion of the region from -500 to -70 caused a 99% reduction in Ren-1 c promoter activity ( Fig. 1 A ). A similar decrease in Ren-1 c promoter activity was also observed for the construct containing a deletion between -197 and -70, indicating the importance of the region between -197 and -70 in regulating the mouse renin gene. Moreover, results with other deletion mutants suggest that sequences from -197 to -157 and from -117 to -70 might contain transcription factor-binding sites contributing to transcriptional activity of this region ( Fig. 1 A ).

Fig. 1. Ren-1 c proximal promoter region from -197 to -70 is essential for mediating Ren-1 c transcriptional activity. Plasmid 4100-based constructs containing the internal deletions ( A ) or constructs with 5'-truncations ( B ) were transfected into As4.1 or JEG-3 cells. Luciferase (Luc) activity is expressed relative to that of plasmid 4100 (arbitrarily set to 100). The HOX·PBX-binding site is shown by a vertical line and indicated by proximal promoter element (PPE). The enhancer is shown by a thicker vertical line. *, : Statistically significant differences ( P < 0.05) relative to the next longer promoter construct in As4.1 and JEG-3 cells, respectively, measured by Student's t -test.

To examine whether the region from -197 to -70 could contribute to basal transcriptional activity without the presence of the enhancer previously identified at -2866 to -2625 ( 22 ), a series of 5'-deletion mutants from -2625 to -59 were transfected into As4.1 ( Fig. 1 B ). Deletion of the sequence from -2625 to -1217 did not significantly alter the expression level, whereas deletion from -1217 to -197 caused a 40% reduction in transcriptional activation. An 81 or 49% decrease in Luc activity was observed when the region from -197 to -94 or -94 to -70, respectively, was removed. Thus the region from -197 to -70 contributes significantly to basal transcriptional activity. Moreover, deletion from -70 to -59, in which the HOX·PBX-binding site is removed, did not cause any significant change in promoter activity, consistent with a previous suggestion that the DNA-bound HOX·PBX·PREP1 complex cannot efficiently activate transcription unless other transcription factor-binding sites are present ( 21, 22 ). To further test whether the region from -197 to -70 in the Ren-1 c promoter regulates renin gene expression in a cell-specific fashion, the same plasmids were transfected into JEG-3 cells, which do not express renin. Results showed that transcriptional activity of 2,625 bp of the Ren-1 c promoter sequence in JEG-3 cells was 14-fold less than that in As4.1 cells. Deletion between -2625 and -1217, -1217 and -197, -94 and -70, or -70 and -59 did not cause any significant change in promoter activity, whereas deletion between -197 and -94 resulted in a 56% decrease in promoter activity, in contrast to the 81% drop in activity for the same deletion assayed in As4.1 cells. These results suggest a cell-specific regulation of Ren-1 c promoter activity by cis -regulatory elements within the region from -197 to -70.

Sequence from -197 to -103 contains two NFI sites and a Sp1/Sp3 site, which are important for Ren-1 c expression. A survey of the sequence from -197 to -70 revealed that two NFI consensus binding sites ( 9 ) and a CACCC site ( 5 ) were present between -197 and -103 ( Fig. 2 ). Similar motifs are located in the enhancer, where they were previously shown to bind NFI and Sp1/Sp3 ( 20 ). To examine whether these elements can bind the predicted transcription factors, EMSAs were performed using double-strand oligonucleotides representing these sites and nuclear extracts prepared from As4.1 cells. Oligonucleotides pRNFIU, pRCACC, and pRNFID containing the putative distal NFI, Sp1/Sp3, and proximal NFI sites and representing the Ren-1 c sequences from -196 to -172, -176 to -155, and -122 to -99 were shown to bind As4.1 cell nuclear proteins ( Fig. 3 ). The DNA·protein complex formed by either pRNFIU or pRNFID could be efficiently competed by 100-fold molar excess of an unlabeled oligonucleotide containing a consensus NFI-binding site (NFI) but not by an oligonucleotide containing a mutated NFI-binding site (mNFI) ( Fig. 3, B and D, lanes 4 and 5 ). Furthermore, both DNA·protein complexes were supershifted by an NFI antibody ( Fig. 3, B and D, lanes 8-10 ). These results indicate that both pRNFIU and pRNFID contain a bona fide NFI-binding site. There is minimal NFI binding to either pRNFIU or pRNFID detected when JEG-3 nuclear extracts were used ( Fig. 3, B and D, lanes 6 and 7 ), consistent with a previous report that JEG-3 cells contain low levels of endogenous NF1 proteins ( 7 ). The presence of a Sp1/Sp3-binding site within oligonucleotide pRCACC was also confirmed by competition and supershift EMSAs. The two complexes (I and II) formed by pRCACC and As4.1 cell nuclear proteins were efficiently competed by pRCACC itself and a Sp1 consensus oligonucleotide (Sp1) but not by mpRCACC or an oligonucleotide containing a mutated Sp1-binding site (mSp1) ( Fig. 3 C, lanes 1-5 ). Similar results were also obtained when JEG-3 nuclear extracts were used in EMSAs ( Fig. 3 C, lanes 6-10 ). Moreover, a Sp1 antibody supershifted complex I, whereas a Sp3 antibody supershifted both complexes I and II ( Fig. 3 C, lanes 11-14 ), suggesting that complex I contains both Sp1 and Sp3, whereas complex II contains an isoform of Sp3 ( 14 ).

Fig. 2. Sequence of the Ren-1 c proximal promoter. The sequence of the Ren-1 c proximal promoter region from -200 to -1 is shown. The previously identified HOX·PBX-binding site and the TATA box are boxed, whereas the transcription factor-binding sites identified in the present study are underlined. The nucleotides involved in the base substitution mutations (mp1-mp9) spanning the region between the Pe site and the TATA box are indicated by overbars, and the altered bases are shown in lower case.

Fig. 3. Identification of 2 nuclear factor I (NFI)-binding sites and an Sp1/Sp3-binding site within the distal portion of the Ren-1 c proximal promoter. A : sequences of oligonucleotides used in EMSAs. Left : oligonucleotides used in NFI identification. Right : oligonucleotides used in Sp1/Sp3 identification. Competition ( left ) and supershift EMSAs ( right ) were performed using pRNFIU ( B ), pRCACC ( C ), and pRNFID ( D ) as labeled probes. Each EMSA uses nuclear extracts prepared from eitehr As4.1 or JEG-3 cells shown with bracketed labels above EMSAs. A 100-fold molar excess of competitor over the probe or 1 µl of each antibody was added in each EMSA reaction. The competitor or antibody added in each lane is indicated above the gels, and the lane without any competitor or antibody added is indicated by -. In lanes labeled with -NFI(-NE), only labeled probe and NFI antiserum were added. The DNA·NFI complex is indicated by an open arrow, whereas the 2 DNA·Sp1/Sp3 complexes are indicated by I and II. The -NFI- and -Sp1-supershifted complexes are labeled SS and SS(Sp1), respectively. FP, free probe.

To test whether the binding of NFI and/or Sp1/Sp3 contributes to transcriptional activity of Ren-1 c, mutations in the NFI- and/or Sp1/Sp3-binding sites were introduced into plasmid 4100 and tested in As4.1 cells for their transcriptional activity ( Fig. 4 ). Mutation of the distal NFI (Pg), Sp1/Sp3 (Pf), or proximal NFI-binding site (Pe) resulted in a 52, 62, or 70% reduction in Ren-1 c promoter activity, respectively, whereas double mutations in Pg and Pf or triple mutations in Pg, Pf, and Pe caused an 85 or 90% loss of Ren-1 c promoter activity, respectively. These results suggest that the NFI- and Sp1/Sp3-binding sites contribute significantly to high-level expression of the mouse renin gene.

Fig. 4. Contribution of the NFI- and Sp1/Sp3-binding sites within the proximal promoter to Ren-1 c transcriptional activity. As4.1 cells were transfected with plasmid 4100-based constructs containing mutations in the NFI- and/or Sp1/Sp3-binding sites. Mutations introduced into Pe, Pf, and Pg in these constructs are the same as in oligonucleotides mpNFID, mpRCACC, and mpNFIU, respectively, used in EMSAs ( Fig. 3 A ). Luc activity is expressed relative to that of plasmid 4100 (arbitrarily set to 100).

Sequence from -103 to -69 contains multiple transcription factor-binding sites. To locate the transcription factor-binding sites 3' to -103 in the proximal promoter of Ren-1 c, a series of mutations containing the indicated base substitutions were made between the proximal NFI site and the TATA box in plasmid 4100 ( Fig. 2 ). Mutations mp4 and mp5 resulted in an 85 and 78% reduction in Ren-1 c promoter 50% reduction in Luc activity, suggesting that these sequences contribute to mouse renin gene expression ( Fig. 5 ).

Fig. 5. Identification of the important cis -regulatory elements in the Ren-1 c sequence from -103 to -30. A series of mutations, mp1-mp9 (see Fig. 2 ), was introduced into the region between the proximal NFI-binding site (Pe) and the TATA box in plasmid 4100. As4.1 cells were transfected with these mutant constructs, and Luc activity was analyzed and is expressed relative to that of plasmid 4100 (arbitrarily set to 100).

To identify the nuclear proteins interacting with these sequences, EMSAs were performed using As4.1 cell nuclear extracts. Binding of nuclear protein to the wild-type oligonucleotides corresponding to those mutated in mp2 could not be detected (data not shown). It is possible that concentration of the protein binding at this site in As4.1 cells is low, the binding requires interaction with other transcription factor-binding sites, or the EMSA condition is not optimized for binding. Alternatively, the DNA structure formed at this site is important for efficient transcription of the Ren-1 c gene. A double-strand oligonucleotide, GC, representing the GC-rich Ren-1 c sequence from -89 to -61, was used in EMSAs to identify the transcription factors binding to nucleotides mutated in mp4 and mp5 ( Fig. 6 A ). Three GC/As4.1 cell nuclear protein complexes (I, II, and III) were observed, which could be efficiently competed by a 100-fold molar excess of unlabeled GC itself ( Fig. 6 B ). To precisely define the nucleotides interacting with the nuclear proteins in these complexes, a series of mutant oligonucleotides (GCm1-GCm6) containing sequential 4-bp mutations were used as competitors in EMSAs. Complex I was efficiently competed by every mutant competitor except GCm5, indicating that the CCTG motif (Pb) is necessary for binding the transcription factors in complex I. Moreover, only GCm3 was incapable of competing for the formation of complex II, suggesting that the CCAC motif (Pc) is sufficient to bind the nuclear proteins in complex II. Finally, complex III could not be efficiently competed by GCm2 and GCm3, demonstrating that the GGTCCCAC motif is able to form complex III with As4.1 cell nuclear proteins. These results are in concordance with those from the mutational analysis in transfection assays ( Fig. 5 ). Similar EMSAs were performed using JEG-3 nuclear extracts ( Fig. 6 C ). Complex II, but not complexes I and III, was present in EMSAs with JEG-3 nuclear extracts, whereas complex IV, which binds similar nucleotide sequence as complex II, was only observed in EMSAs with JEG-3 nuclear extracts.

Fig. 6. Identification of the CCAC and CCTG motifs immediately 5' to the HOX·PBX-binding site as the transcription factor-binding sites. A : sequences of oligonucleotides used in EMSAs. EMSAs were performed using GC as the labeled probe and mutated GCs (GCm1-GCm6), which contain sequential 4-bp mutations, as competitors (100 x ) to identify the nucleotides in GC necessary for the binding of nuclear proteins from either As4.1 ( B ) or JEG-3 cells ( C; bracketed labels above EMSAs). In C, shifted complexes from EMSAs with As4.1 nuclear extracts ( left ) and shifted complexes from EMSAs with JEG-3 nuclear extracts ( right ) are labeled. Three specific GC/As4.1 nuclear protein complexes (I, II, and III) and 2 specific GC/JEG-3 nuclear protein complexes (II and IV) were identified. NS, nonspecific DNA·protein complex. Free probe is not shown.

Because the mutation mp9 also decreased Ren-1 c transcriptional activity ( Fig. 5 ), we investigated whether these mutated nucleotides were also involved in the binding of As4.1 cell nuclear proteins. Oligonucleotide Pd representing the Ren-1 c sequence from -109 to -90 was used in EMSAs with competitors containing sequential 3- or 4-bp mutations ( Fig. 7 A ). A single complex was formed with Pd and As4.1 cell nuclearproteins, which could be efficiently competed by Pd itself, Pdm1, pdm2, pdm4, and pdm6 but not Pdm3 and Pdm5 ( Fig. 7 B ). These results suggest that the AAAANNNGCT motif (Pd) binds transcription factors and contributes to high-level Ren-1 c expression in As4.1 cells. A similar transcription factor, which is slightly larger and binds the AAAANNNGCT motif, is also present in JEG-3 cells ( Fig. 7 C ).

Fig. 7. Identification of nuclear proteins binding to Pd. A : sequences of oligonucleotides used in EMSAs. EMSAs were performed using Pd as the labeled probe and mutated Pds (Pdm1-Pdm6), which contain sequential 3- or 4-bp mutations, as competitors (100 x ). Either As4.1 ( B ) or JEG-3 nuclear extracts ( C; bracketed labels above EMSAs) were used. In C, shifted complexes from EMSAs with As4.1 nuclear extracts ( left ) and shifted complexes from EMSAs with with JEG-3 nuclear extracts ( right ) are labeled. The specific Pd/nuclear protein complex (open arrow) was observed in EMSAs with either As4.1 or JEG-3 nuclear extracts.

Ren-1 c expression is dependent on the functional cooperation among the transcription factors binding to the proximal promoter. To better understand the roles of transcription factors binding to the proximal promoter in Ren-1 c expression, mutations were introduced into two or more of these protein-binding sites simultaneously in plasmid 4100 ( Fig. 8 ). Mutations of both Pb and Pc did not cause a greater reduction in Ren-1 c expression than a single mutation in either Pb or Pc ( Figs. 5 and 8 ), implicating that proteins binding to Pb and Pc may interact with each other to contribute to Ren-1 c expression. Mutations of both Pa (the HOX·PBX binding site) and Pc or all of Pa, Pb, and Pc sites resulted in a 98% reduction in Ren-1 c promoter activity ( Fig. 8 ), suggesting that the HOX·PBX site functionally cooperates with the Pb and/or Pc sites to regulate Ren-1 c expression. Results from double mutations in Pe (the proximal NFI-binding site) and Pd also suggest a functional cooperation between these two sites. Moreover, mutations of all the transcription factor-binding sites 5' to the HOX·PBX site caused a 99% decrease in Ren-1 c promoter activity, which is equal to the percent drop when the sequence from -197 to -70 was deleted from plasmid 4100 ( Fig. 1 A ). This suggests that all the important cis -acting regulatory elements in this region have been identified. Finally, mutations of all the identified transcription factor-binding sites binding to the proximal promoter resulted in a Ren-1 c promoter with only 0.24% of wild-type promoter activity, demonstrating the pivotal role of the proximal promoter in regulating the mouse renin gene.

Fig. 8. Mutational analysis of transcription factor-binding sites within the Ren-1 c proximal promoter. As4.1 cells were transfected with plasmid 4100-based constructs containing mutations in the transcription factor-binding sites. Mutations introduced into Pa, Pb, Pc, and Pd in these constructs are same as in constructs d ( Fig. 4 in Ref. 22 ), 4100mp4, 4100mp5, and 4100mp9 ( Fig. 5 ), respectively. Luc activity is expressed relative to that of plasmid 4100 (arbitrarily set to 100).

Roles of the proximal promoter elements and enhancer in ET-1-induced inhibition of Ren-1 c expression. It has been reported that treatment of As4.1 cells with ET-1 results in 68% reduction in renin mRNA level, and this reduction is caused by inhibition of renin gene transcription rather than changes in message decay ( 26 ). To examine whether the transcription factor-binding sites (Pa-Pg) within the Ren-1 c proximal promoter contribute to ET-1-induced inhibition, plasmid 4100- and 4100-based constructs containing mutations in these sites were transfected into As4.1 cells and either left untreated or treated with ET-1 for 24 h. Treatment with ET-1 caused a 56% reduction in transcriptional activity of 4.1 kb of the Ren-1 c 5'-flanking sequence ( Fig. 9 A ). Mutations of all Pd, Pe, Pf, and Pg did not significantly change the ET-1 effect, whereas mutation of the HOX·PBX-binding site (Pa), Pb, Pc, both Pb and Pc, or all Pb, Pc, Pd, Pe, Pf, and Pg caused a small but significant reduction in the ET-1 inhibitory effect. Mutations of all Pa, Pb, and Pc or all the transcription factor-binding sites identified within the proximal promoter resulted in a 30 or 27% decrease in the ET-1 inhibitory effect, respectively. However, when construct 2625 or 197 was tested, no ET-1 inhibitory effect was observed. These results suggest that Pa, Pb, and Pc are required for the maximal ET-1 inhibitory effect, but the proximal promoter may not be the direct target of ET-1 modification. These results also suggest that the enhancer located at -2866 to -2625 is a likely target of ET-1 action. Plasmid Enh-TA, which contains the Ren-1 c enhancer inserted immediately upstream of an E1b TATA box, was tested in As4.1 cells for the ET-1 effect ( Fig. 9 B ). ET-1 inhibited transcriptional activity induced by the 242-bp Ren-1 c enhancer by 36%, whereas ET-1 does not have any inhibitory effect on transcriptional activity induced by the E1b TATA box alone, demonstrating that the Ren-1 c enhancer is a direct target of the ET-1-induced signal pathway.

Fig. 9. Roles of the proximal promoter and enhancer in endothelin-1 (ET-1)-induced inhibition of Ren-1 c transcription. A : As4.1 cells were transfected with plasmid 4100, 4100-based constructs containing mutations in proximal promoter elements, and constructs containing Ren-1 c promoter without the presence of the enhancer and either left untreated or treated with 10 nM ET-1 for 24 h. Effect of ET-1 (%) is expressed as a percentage of [(Luc activity from ET-1-treated cells/Luc activity from untreated cells) x 100]. Basal transcriptional activities for these constructs are shown in Figs. 1 B and 8. *Significantly different ( P < 0.05) ET-1 effect for the construct from that of construct 4100 as measured by Student's t -test. B : As4.1 cells were transfected with plasmids Enh-TA containing the Ren-1 c enhancer inserted immediately upstream of the adenovirus E1b TATA box and TA containing only the E1b TATA box and either left untreated or treated with ET-1 for 24 h. Luc activity is expressed relative to that of plasmid 4100 (arbitrarily set to 100). *Significantly different ( P < 0.05) as measured by Student's t -test.

DISCUSSION

A novel region (-197 to -69) has been identified in the proximal promoter of the Ren-1 c gene, which consists of at least six transcription factor-binding sites and is critical for Ren-1 c expression. Two NFI- and a Sp1/Sp3-binding site were found within the distal portion of the region, mutations of which resulted in a 90% decrease in Ren-1 c promoter activity. Meanwhile, three other transcription factor-binding sites, including Pb, Pc, and Pd, were found between the proximal NFI site and the HOX·PBX-binding site. Mutation of Pd caused a 60% reduction in Ren-1 c transcriptional activation, whereas mutation of Pb or Pc reduced transcriptional activity by 78 or 85%, respectively. Moreover, mutations of all six sites decreased Ren-1 c transcriptional activation by 99%. Finally, mutations of all six newly identified sites plus the HOX·PBX site reduced Ren-1 c transcriptional activity by 99.8%. These results suggest a pivotal role for the proximal promoter region in transcriptional regulation of the mouse renin genes.

Identification of the transcription factor-binding sites other than the HOX·PBX-binding site in the proximal promoters of the renin genes has been reported. Tamura et al. ( 36 ) reported the requirement of two elements, RU-1 (-224 to -138) and RP-2 (-75 to -47), for the detected promoter activity of Ren-1 c in embryonic kidney-derived 293 cells. However, the transcription factors binding to these elements were not identified. Because 293 cells are not renin-expressing cells, the transcription factors binding to these regions may not be the same as those in As4.1 cells. Pinet and co-workers ( 6, 15 ) also reported the identification of several transcription factor-binding sites, including a cAMP-responsive element, an AGE-3-like sequence, an apolipoprotein A1 regulatory protein-1-like sequence, and a sequence homologous to the ETS-binding site in the human renin promoter (-300 to -1) in renin-producing cells derived from chorionic tissues. It seems that these transcription factor-binding sites are different from the Pb (CCTG), Pc (CCAC), and Pd motifs in the mouse promoter. However, a survey of nucleotide sequences in human and rat proximal promoter regions ( 21 ) revealed the presence of the CCTG motif in both human and rat renin promoters. Whether the CCTG motif contributes to human or rat renin gene expression remains to be studied.

Six NFI-binding sites have been found in the 5'-flanking sequence of the Ren-1 c gene, with four in the distal portion of the enhancer and two in the proximal promoter. Also, in both the enhancer and the proximal promoter, a Sp1/Sp3-binding site is found next to the NFI site. Moreover, these two sites are capable of cooperating with each other. The physical interaction between NFI-X, which is the predominant NFI isoform in As4.1 cells ( 20 ), and Sp1 has been reported ( 23 ). NFI-X is able to repress PDGF A-chain transcription by directly interacting with Sp1 and antagonizing Sp1 occupancy of the promoter. Whether these NFI/Sp sites have any role in regulating renin expression other than simply providing for basal transcriptional activity remains to be determined.

Transfections of As4.1 and JEG-3 cells with truncated Ren-1 c promoter constructs showed that the region from -197 to -70 was distinctly regulated in these two different cell lines ( Fig. 1 B ). EMSAs were then performed to further address the possibility that the distinct regulation is caused by binding of different transcription factors to cis -regulatory elements within the region ( Figs. 3, 6, and 7 ). We showed that both cell lines contain Sp1/Sp3- and Pd-binding proteins, whereas in JEG-3 cells NFI- and Pb-binding proteins (complexes I and III, Fig. 6 ) are absent. Moreover, JEG-3 cells contain additional proteins (complex IV, Fig. 6 C ) binding to Pc compared with As4.1 cells. Results from EMSAs are in good agreement with data from transfection assays, demonstrating that the region from -197 to -70 contains cis -regulatory elements contributing to cell-specific regulation of Ren-1 c expression.

The HOX·PBX-binding site, Pb, and Pc are the more important sites within the proximal promoter. Mutation of each individual site caused a 93, 78, and 85% decrease in Ren-1 c transcriptional activity, respectively ( Figs. 5 and 8 ). Mutations of all three 98%. The identities of transcription factors binding to either Pb or Pc are still unknown. The GC-rich sequence (-89 to -61) spanning these two sites contains elements resembling binding sites for transcription factors such as AP2 ( 10 ), Cbfa1 ( 8 ), and CP2 ( 41 ). However, the competition EMSAs showed that none of these factors bind to this region (data not shown). Yeast one-hybrid experiments are being performed to identify the factors binding to the CCTG (Pb) and CCAC (Pc) motifs. Presently, little is known about the transcription factors that can interact or cooperate with the HOX·PBX·PREP1/MEIS complex in HOX target genes. The identification of these factors will shed light on the mechanism of HOX action and tissue-specific and developmental regulation of the mouse renin genes.

Using the As4.1 cell line as a model system, we have identified two important control regions, the enhancer (-2866 to -2625) and the proximal promoter region (-197 to -50), in the mouse Ren-1 c 5'-flanking sequence. Each of the control regions contains numerous transcription factor-binding sites important for Ren-1 c expression. However, how these two regions communicate with each other to regulate renin gene expression remains to be studied. Luc activity from the 4100-based construct containing mutations of all the transcription factor-binding sites within the proximal promoter region is only threefold more than that from construct 59, which contains only the minimal Ren-1 c promoter (-59 to +6), demonstrating that the enhancer function is severely impaired when all the identified transcription factor-binding sites in the proximal promoter are mutated ( Figs. 1 B and 7 ). This indicates that the function of the enhancer is dependent on the presence of the constellation of proximal promoter elements. Identification of all the transcription factors binding to the enhancer and the proximal promoter region will facilitate the reconstitution of the renin transcription system in non-renin-producing cell lines (e.g.. Ltk-) and further our understanding of the mechanisms of transcriptional regulation of the mouse renin genes.

ET-1, a potent vasoconstrictor originally identified in vascular endothelial cells ( 40 ), has been shown to inhibit cAMP-stimulated renin production and release in juxtaglomerular cells ( 3, 24 ) and attenuates renin gene expression in As4.1 cells ( 26 ). The effects of ETs (ET-1, ET-2, and ET-3) are mediated by two distinct receptors (ET A R and ET B R) coupled to G proteins (see Refs. 25 and 34 for reviews). Binding of ETs to ETRs results in activation of phosphoinositide-specific phospholipase C, calcium flux, activation of protein kinase C, inhibition of cAMP production, and activation of different mitogen-activated protein kinase-dependent signaling pathways. We have shown that the transcription factor-binding sites in the Ren-1 c proximal promoter, especially Pa, Pb, and Pc, are required for the maximal inhibition by ET-1. However, the Ren-1 c proximal promoter does not seem to be a direct target of ET-1 action because neither construct 2625 nor 197, both of which contain the identified proximal promoter transcription factor-binding sites, responds to ET-1 with reduced transcriptional activity. On the other hand, the Ren-1 c enhancer has been shown to be the target sequence for ET-1 inhibition, which is also the target for other signaling molecules, including cAMP ( 19 ), retinoic acid ( 29 ), and inflammatory cytokines (37; Pan L, Wang Y, Jones CA, Glenn ST, Baumann H, and Gross KW, unpublished observations). A possible reason for involvement of cis -regulatory elements within the proximal promoter in ET-1 inhibition of Ren-1 c expression is that these sites are important for the interaction with the enhancer, and mutations in these sites result in reduced interaction with the enhancer and, consequently, decreased ET-1 inhibition.

GRANTS

This work was supported by National Institutes of Health (NIH) Grant HL-48459 (to K. W. Gross) and funds from the Bruce Cuvelier Family. L. Pan was supported by a postdoctoral fellowship from NIH. This research utilized core facilities supported in part by RPCI NCI-funded Cancer Center Support Grant CA-16056.

ACKNOWLEDGMENTS

We thank Dr. Naoko Tanese for the gift of NFI antiserum.

【参考文献】
  Abel KJ, Howles PN, and Gross KW. DNA insertions distinguish the duplicated renin genes of DBA/2 and M. hortulanus mice. Mamm Genome 2: 32-40, 1992.

Abonia JP, Howles PN, Abel KJ, Black TA, Jones CA, and Gross KW. Evaluating a model of an NRE mediated tissue-specific expression of murine renin genes. Hypertension 37: 105-109, 2001.

Ackermann M, Ritthaler T, Riegger G, Kurtz A, and Kramer BK. Endothelin inhibits cAMP-induced renin release from isolated renal juxtaglomerular cells. J Cardiovasc Pharmacol 26, Suppl 3: S135-S137, 1995.

Barrett G, Horiuchi M, Paul M, Pratt RE, Nakamura N, and Dzau VJ. Identification of a negative regulatory element involved in tissue-specific expression of mouse renin genes. Proc Natl Acad Sci USA 89: 885-889, 1992.

Black AR, Black JD, and Azizkhan-Clifford J. Sp1 and Kruppel-like factor family of transcription factors in cell growth regulation and cancer. J Cell Physiol 188: 143-160, 2001.

Borensztein P, Germain S, Fuchs S, Philippe J, Corvol P, and Pinet F. Cis -regulatory elements and trans -acting factors directing basal and cAMP-stimulated human renin gene expression in chrorionic cells. Circ Res 74: 764-773, 1994.

Bruggemeier U, Rogge L, Winnacker EL, and Beato M. Nuclear factor I acts as a transcription factor on the MMTV promoter but competes with steroid hormone receptors for DNA binding. EMBO J 9: 2233-2239, 1990.

Ducy P. Cbfa1: a molecular switch in osteoblast biology. Dev Dyn 219: 461-471, 2000. <a href="/cgi/external_ref?access_num=10.1002/1097-0177(2000)9999:9999

Gronostajski RM. Roles of the NFI/CTF gene family in transcription and development. Gene 249: 31-45, 2000.

Hilger-Eversheim K, Moser M, Schorle H, and Buettner R. Regulatory roles of AP-2 transcription factors in vertebrate development, apoptosis and cell-cycle control. Gene 260: 1-12, 2000.

Horiuchi M, Pratt RE, Nakamura N, and Dzau VJ. Distinct nuclear proteins competing for an overlapping sequence of cyclic adenosine monophosphate and negative regulatory elements regulate tissue-specific mouse renin gene expression. J Clin Invest 92: 1805-1811, 1993.

Jones CA, Fabian JR, Abel KJ, Sigmund CD, and Gross KW. The regulation of renal and extrarenal renin gene expression in the mouse. In: Cellular and Molecular Biology of the Renin-Angiotensin System, edited by Raizada MK, Phillips MI, and Sumners C. Boca Raton, FL: CRC, 1993, p. 33-57.

Jones CA, Petrovic N, Novak EK, Swank RT, Sigmund CD, and Gross KW. Biosynthesis of renin in mouse kidney tumor As4.1 cells. Eur J Biochem 243: 181-190, 1997.

Kennett SB, Udvadia AJ, and Horowitz JM. Sp3 encodes multiple proteins that differ in their capacity to stimulate or repress transcription. Nucleic Acids Res 25: 3110-3117, 1997.

Konoshita T, Germain S, Philippe J, Corvol P, and Pinet F. Evidence that renal and chotionic tissues contain similar nuclear binding proteins that recognize the human renin promoter. Kidney Int 50: 1515-1524, 1996.

Li YC, Kong J, Wei M, Chen ZF, Liu SQ, and Cao LP. 1,25-Dihydroxyvitamin D 3 is a negative endocrine regulator of the renin-angiotensin system. J Clin Invest 110: 229-238, 2002.

Liu X, Huang X, and Sigmund CD. Identification of a nuclear orphan receptor (Ear2) as a negative regulator of renin gene transcription. Circ Res 92: 1033-1040, 2003.

Nakamura N, Burt DW, Paul M, and Dzau VJ. Negative control elements and cAMP responsive sequences in the tissue-specific expression of mouse renin genes. Proc Natl Acad Sci USA 86: 56-59, 1989.

Pan L, Black TA, Shi Q, Jones CA, Petrovic N, Loudon J, Kane C, Sigmund CD, and Gross KW. Critical roles of a cyclic AMP responsive element and an E-box in regulation of mouse renin gene expression. J Biol Chem 276: 45530-45538, 2001.

Pan L, Glenn ST, Jones CA, Gronostajski RM, and Gross KW. Regulation of renin enhancer activity by nuclear factor I and Sp1/Sp3. Biochim Biophys Acta 1625: 280-290, 2003.

Pan L, Xie Y, Black TA, Jones CA, Pruitt SC, and Gross KW. An Abd-B class HOX·PBX recognition sequence is required for expression from the mouse Ren-1 c gene. J Biol Chem 276: 32489-32494, 2001.

Petrovic N, Black TA, Fabian JR, Kane C, Jones CA, Loudon JA, Abonia JP, Sigmund CD, and Gross KW. Role of proximal promoter elements in regulation of renin gene transcription. J Biol Chem 271: 22499-22505, 1996.

Rafty LA, Santiago FS, and Khachigian LM. NF1/X represses PDGF A-chain transcription by interacting with Sp1 and antagonizing Sp1 occupancy of the promoter. EMBO J 21: 334-343, 2002.

Ritthaler T, Scholz H, Ackermann M, Riegger G, Kurtz A, and Kramer BK. Effects of endothelins on renin secretion from isolated mouse renal juxtaglomerular cells. Am J Physiol Renal Fluid Electrolyte Physiol 268: F39-F45, 1995.

Rubanyi GM and Polokoff MA. Endothelins: molecular biology, biochemistry, pharmacology, physiology, and pathophysiology. Pharmacol Rev 46: 325-415, 1994.

Ryan MJ, Black TA, Millard SL, Gross KW, and Hajduczok G. Endothelin-1 increases calcium and attenuates renin gene expression in As4.1 cells. Am J Physiol Heart Circ Physiol 283: H2458-H2465, 2002.

Santoro C, Mermod N, Andrews PC, and Tjian R. A family of human CCAAT-box-binding proteins active in transcription and DNA replication: cloning and expression of multiple cDNAs. Nature 334: 218-224, 1988.

Shi Q, Black TA, Gross KW, and Sigmund CD. Species-specific differences in positive and negative regulatory elements in the renin gene enhancer. Circ Res 85: 479-488, 1999.

Shi Q, Gross KW, and Sigmund CD. Retinoic acid-mediated activation of the mouse renin enhancer. J Biol Chem 276: 3597-3603, 2001.

Shi Q, Gross KW, and Sigmund CD. NF-Y antagonizes renin enhancer function by blocking stimulatory transcription factors. Hypertention 38: 332-336, 2001.

Sigmund CD. Regulation of renn expression and blood pressure by vitamin D 3. J Clin Invest 110: 155-156, 2002.

Sigmund CD and Gross KW. Structure, expression and regulation of murine renin genes. Hypertension 18: 446-457, 1991.

Sigmund CD, Okuyama K, Ingelfinger J, Jones CA, Mullins JJ, Kane C, Kim U, Wu C, Kenny L, Rustum Y, Dzau VJ, and Gross KW. Isolation and characterization of renin expressing cell lines from transgenic mice containing a renin-promoter viral oncogene fusion construct. J Biol Chem 265: 19916-19922, 1990.

Sokolovsky M. Endothelin receptor subtypes and their role in transmembrane signaling mechanisms. Pharmacol Ther 68: 435-471, 1995.

Tamura K, Chen YE, Horiuchi M, Chen Q, Daviet L, Yang Z, Lopez-Ilasaca M, Mu H, Pratt RE, and Dzau VJ. LXR functions as a cAMP-responsive transcriptional regulator of gene expression. Proc Natl Acad Sci USA 97: 8513-8518, 2000.

Tamura K, Tanimoto K, Murakami K, and Fukamizu A. A combination of upstream and proximal elements is required for efficient expression of the mouse renin promoter in cultured cells. Nucleic Acids Res 20: 3617-3623, 1992.

Todorov VT, Volkl S, Muller M, Bohla A, Klar J, Kunz-Schughart LA, Hehlgans T, and Kurtz A. TNF- activates NF- B to inhibit renin transcription by targeting cAMP responsive element. J Biol Chem 279: 1458-1467, 2003.

Yamada T, Horiuchi M, Morishita R, Zhang L, Pratt RE, and Dzau VJ. In vivo indentification of a negative regulatory element in the mouse renin gene using direct gene transfer. J Clin Invest 96: 1230-1237, 1995.

Yan Y, Jones CA, Sigmund CD, Gross KW, and Catanzaro DF. Conserved enhancer elements in human and mouse renin genes have different transcriptional effects in As4.1 cells. Circ Res 81: 558-566, 1997.

Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Yazaki Y, Goto K, and Masaki Y. A novel potent vasoconstrictor peptide producing vascular endothelial cells. Nature 332: 411-415, 1988.

Yoon JB, Li G, and Roeder RG. Characterization of a family of related cellular transcription factors which can modulate human immunodeficiency virus type 1 transcription in vitro. Mol Cell Biol 14: 1776-1785, 1994.

作者单位：Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, New York 14263-0001