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Activation of the Rat Renin Promoter by HOXD10·PBX1b·PREP1, Ets-1, and the Intracellular Domain of Notch

2005; Elsevier BV; Volume: 280; Issue: 21 Linguagem: Inglês

10.1074/jbc.m414618200

1083-351X

Pan Li, Sean T. Glenn, Craig A. Jones, Kenneth W. Gross,

RNA modifications and cancer

Renin gene expression is subject to complex developmental and tissue-specific regulation. A comparison of the promoter sequences of the human, rat, and mouse renin genes has revealed a highly conserved sequence homologous to the DNA recognition sequence for CBF1 (CSL/RBP-Jκ/Su(H)/LAG1/RBPSUH). Electrophoretic mobility shift assays document that As4.1 cell nuclear protein complex binding to the putative rat renin CBF1-binding site (-175 to -168 bp) contains CBF1. Transient transfection analyses in COS-7 cells further document that a CBF1-VP16 fusion protein and the intracellular domain of Notch1 robustly activate a promoter containing multiple copies of the rat renin CBF1-binding site. An Ets-binding site (-143 to -138 bp) has also been identified in the rat renin promoter by sequence comparisons and electrophoretic mobility shift assays. Transcription factor Ets-1 is capable of activating the rat renin promoter through the Ets-binding site. Mutation of the CBF-binding site significantly increases transcriptional activity of the rat renin promoter in Calu-6 and COS-7 cells but not in As4.1 cells, whereas mutation of the Ets-binding site reduces promoter activity of the rat renin gene in all three cell lines. Finally, we show that the intracellular domain of Notch1, Ets-1, and HOXD10·PBX1b·PREP1 activate the rat renin promoter cooperatively in COS-7 cells. These results strongly suggest that the renin gene is a downstream target of the Notch signaling pathway. Renin gene expression is subject to complex developmental and tissue-specific regulation. A comparison of the promoter sequences of the human, rat, and mouse renin genes has revealed a highly conserved sequence homologous to the DNA recognition sequence for CBF1 (CSL/RBP-Jκ/Su(H)/LAG1/RBPSUH). Electrophoretic mobility shift assays document that As4.1 cell nuclear protein complex binding to the putative rat renin CBF1-binding site (-175 to -168 bp) contains CBF1. Transient transfection analyses in COS-7 cells further document that a CBF1-VP16 fusion protein and the intracellular domain of Notch1 robustly activate a promoter containing multiple copies of the rat renin CBF1-binding site. An Ets-binding site (-143 to -138 bp) has also been identified in the rat renin promoter by sequence comparisons and electrophoretic mobility shift assays. Transcription factor Ets-1 is capable of activating the rat renin promoter through the Ets-binding site. Mutation of the CBF-binding site significantly increases transcriptional activity of the rat renin promoter in Calu-6 and COS-7 cells but not in As4.1 cells, whereas mutation of the Ets-binding site reduces promoter activity of the rat renin gene in all three cell lines. Finally, we show that the intracellular domain of Notch1, Ets-1, and HOXD10·PBX1b·PREP1 activate the rat renin promoter cooperatively in COS-7 cells. These results strongly suggest that the renin gene is a downstream target of the Notch signaling pathway. Renin, through its participation in an enzymatic cascade that results in the production of angiotensin II, the major effector molecule of the renin-angiotensin system, plays a major role in blood pressure regulation and electrolyte homeostasis. Expression of the renin gene is subject to complex tissue-specific and developmental regulation. Progress has been made recently in understanding the mechanisms of this regulation (1Pan L. Gross K.W. Hypertension. 2005; 45: 3-8Crossref PubMed Scopus (73) Google Scholar). An enhancer (2Petrovic N. Black T.A. Fabian J.R. Kane C. Jones C.A. Loudon J.A. Abonia J.P. Sigmund C.D. Gross K.W. J. Biol. Chem. 1996; 271: 22499-22505Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 3Pan L. Black T.A. Shi Q. Jones C.A. Petrovic N. Loudon J. Kane C. Sigmund C.D. Gross K.W. J. Biol. Chem. 2001; 276: 45530-45538Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar) and a proximal promoter region (4Pan L. Jones C.A. Glenn S.T. Gross K.W. Am. J. Physiol. 2004; 286: F1107-F1115Google Scholar) have been identified as critical for expression of the mouse renin gene (Ren-1c) in the mouse kidney tumor-derived As4.1 cell line. This cell line was developed from a Ren-2-T antigen transgenic line and has been shown to retain many features characteristic of renin-expressing juxtaglomerular cells in the kidney (5Sigmund C.D. Okuyama K. Ingelfinger J. Jones C.A. Mullins J.J. Kane C.M. Kim U. Wu C. Kenny L. Rustum Y. Dzau V.J. Gross K.W. J. Biol. Chem. 1990; 265: 19916-19922Abstract Full Text PDF PubMed Google Scholar). A HOX·PBX-binding site has been located within the Ren-1c proximal promoter region (6Pan L. Xie Y. Black T.A. Jones C.A. Pruitt S.C. Gross K.W. J. Biol. Chem. 2001; 276: 32489-32494Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Mutation of the site in a construct containing 4.1 kb of the Ren-1c 5′-flanking sequence caused a more than 10-fold decrease in transcriptional activity. HOXD10 is capable of pairing with PBX1b and binding to the Ren-1c HOX·PBX site with high affinity. Moreover, PREP1 has been shown to form a ternary complex with HOXD10 and PBX1b on the Ren-1c promoter. The HOX·PBX-binding site is also present in human and rat renin promoters, suggesting an important role of this site in the regulation of renin expression. Notch is a transmembrane receptor that regulates expression of genes in a cell type-specific fashion to determine cell fate and patterning through cell-cell communication (see Refs. 7Lai E.C. Development (Camb.). 2004; 131: 965-973Crossref PubMed Scopus (840) Google Scholar, 8Kopan R. J. Cell Sci. 2002; 115: 1095-1097Crossref PubMed Google Scholar, 9Schweisguth F. Curr. Biol. 2004; 14: R129-R138Abstract Full Text Full Text PDF PubMed Google Scholar for reviews). There are four Notch genes (Notch1–Notch4) in mammals. Upon receipt of extracellular signals mediated via binding of the specific ligands, Jagged and Delta-like, the intracellular domain of Notch is released by proteolytic cleavages and translocates to the nucleus, where it subsequently interacts with the transcriptional repressor CBF1 and converts it to a transcriptional activator through replacement of the CBF1-bound co-repressor complex with a co-activator complex. Several co-repressors have been identified, including CIR, SMART, and N-CoR (10Hsieh J.J. Zhou S. Chen L. Young D.B. Hayward S.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 23-28Crossref PubMed Scopus (250) Google Scholar, 11Kao H.Y. Ordentlich P. Koyano-Nakagawa N. Tang Z. Downes M. Kintner C.R. Evans R.M. Kadesch T. Genes Dev. 1998; 12: 2269-2277Crossref PubMed Scopus (486) Google Scholar), whereas Mastermind appears to be a major co-activator for Notch signaling (12Petcherski A.G. Kimble J. Nature. 2000; 405: 364-368Crossref PubMed Scopus (155) Google Scholar, 13Wu L. Aster J.C. Blacklow S.C. Lake R. Artavanis-Tsakonas S. Griffin J.D. Nat. Genet. 2000; 26: 484-489Crossref PubMed Scopus (458) Google Scholar). In this study, we identified two new transcription factor-binding sites, a CBF1- and an Ets-binding site, in the promoter region of the rat renin gene in addition to the HOX·PBX-binding site identified previously (6Pan L. Xie Y. Black T.A. Jones C.A. Pruitt S.C. Gross K.W. J. Biol. Chem. 2001; 276: 32489-32494Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The CBF1-binding site acts as a negative regulatory element in renin-expressing Calu-6 cells and non-renin-expressing COS-7 cells, whereas the Ets-binding site is a positive regulatory element in all cell lines tested. Moreover, we showed that N1IC 1The abbreviations used are: N1IC, intracellular domain of Notch1; N3IC, intracellular domain of Notch3; EMSA, electrophoretic mobility shift assay; CMV, cytomegalovirus; RSV, Rous sarcoma virus. 1The abbreviations used are: N1IC, intracellular domain of Notch1; N3IC, intracellular domain of Notch3; EMSA, electrophoretic mobility shift assay; CMV, cytomegalovirus; RSV, Rous sarcoma virus. and Ets-1 activated the rat renin promoter through the CBF1- and Ets-binding sites, respectively. Finally, we demonstrated that N1IC, Ets-1, and HOXD10·PBX1b·PREP1 were capable of cooperating with each other to activate the rat renin promoter. Plasmids—Plasmid rR240 was constructed by inserting a fragment containing the rat renin gene region from -241 to +16, which was amplified by the polymerase chain reaction from a bacterial artificial chromosome, CH230 101 J7, containing the rat renin genomic sequence, into the XhoI/HindIII-digested pGL2-basic (Promega). Plasmids containing mutations in the transcription factor-binding sites of the rat renin promoter (see Fig. 3) were created using the QuikChange site-directed mutagenesis kit (Stratagene). The oligonucleotides used to generate mutations in the CBF1-, Ets-, and HOX·PBX-binding site are 5′-CTGGGTTCAGCCATGTTTaaagCACTCGATTCCTGCCACTC-3′, 5′-CCTGCCACTCTGCTTCGCTTaaGGCTCCTGCTTATCCCTC-3′, and 5′-GGACCCTGGGGTAAccAActAGAGCAGGGCCTG-3′, respectively. Plasmid 7XrCBF1-SV40 or 5XmrCBF1-SV40 was constructed by inserting seven tandem copies of a double-stranded oligonucleotide containing the rat renin CBF1-binding site (5′-tcgagGCCATGTTTCCCACACTCGATTc-3′) or five tandem copies of an oligonucleotide containing the mutated CBF1-binding site (5′-tcgagGCCATGTTTaaagCACTCGATTc-3′), respectively, into the XhoI site of pGL2-Promoter (Promega). The expression vectors for Ets-1, Ets-2, and CBF1 contain the I.M.A.G.E. full-length cDNA clones inserted in pCMV·SPORT6 and were purchased from Open Biosystems. The mouse N1IC expression plasmid contains the intracellular domain of Notch1 under the control of a CMV promoter and was kindly provided by Dr. R. Kopan (Washington University School of Medicine, St Louis, MO) (14Kopan R. Nye J.S. Weintraub H. Development. 1994; 120: 2385-2396Crossref PubMed Google Scholar). VP16-CBF1 was constructed by inserting the polymerase chain reaction-amplified cDNA encoding CBF1 into the EcoRI/XbaI-digested pVP16 (BD Biosciences). The expression vectors for HOXD10, PBX1b, and PREP1 contain the mouse full-length cDNAs inserted in pcDNA3.1/myc-His(+)A and were described previously (6Pan L. Xie Y. Black T.A. Jones C.A. Pruitt S.C. Gross K.W. J. Biol. Chem. 2001; 276: 32489-32494Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Cell Culture and Transient Transfections—As4.1 and COS-7 cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Calu-6 cells were grown in Eagle's minimal essential medium supplemented with non-essential amino acids and 10% fetal bovine serum. All three of these cell lines were transiently transfected using Lipofectamine (Invitrogen). For each transfection in a 35-mm culture dish, ∼2 μg of DNA (see figure legends for the amount of each plasmid added in a transfection assay) were mixed with 6 μl of Lipofectamine. Forty-eight h after transfection, cells were harvested and measured for luciferase and β-galactosidase activity using the luciferase assay system (Promega) and Galacto-Light Plus chemiluminescent reporter assay (Tropix), respectively. Luciferase activity is normalized with β-galactosidase activity to correct for differences in transfection efficiency between experiments. Results are expressed as the mean ± S.E. of at least three separate experiments. EMSA—The EMSA was performed as described previously (3Pan L. Black T.A. Shi Q. Jones C.A. Petrovic N. Loudon J. Kane C. Sigmund C.D. Gross K.W. J. Biol. Chem. 2001; 276: 45530-45538Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). The CBF1 antiserum (K-0043) (15Hamaguchi Y. Yamamoto Y. Iwanari H. Maruyama S. Furukawa T. Matsunami N. Honjo T. J. Biochem. 1992; 112: 314-320Crossref PubMed Scopus (60) Google Scholar) used in supershift assay was purchased from the Institute of Immunology, Tokyo, Japan. Identification of a CBF1-binding Site in the Rat Renin Promoter—A phylogenetic footprinting comparison of human, rat, and mouse renin promoter sequences identifies a highly conserved sequence consisting of a TTCCCACA motif (Fig. 1A). Further analysis using the transcription factor database Mat-Inspector suggests that it is a putative binding site for the transcriptional repressor, CBF1. Results from EMSA showed that a double-stranded oligonucleotide, rRen, which represents the rat promoter sequence from -185 to -166 bp containing putative CBF1-binding site, formed a complex with nuclear proteins prepared from As4.1 cells (Fig. 1B). This complex could be efficiently competed by 100-fold molar excess of unlabeled rRen itself, mRen, a corresponding mouse renin promoter sequence, and oligonucleotides containing previously identified CBF1-binding sites, including 23/24, HES-1, NF-kB, and IL-6kB, whereas it could not be competed by oligonucleotides that do not contain the CBF1-binding site such as IL-2kB and IgkB (Fig. 1, A and B) (16Palmieri M. Sasso M.P. Monese R. Merola M. Faggioli L. Tovey M. Furia A. Nucleic Acids Res. 1999; 27: 2785-2791Crossref PubMed Scopus (33) Google Scholar). Furthermore, a CBF1-specific antibody supershifted the DNA·protein complex. These results demonstrate that the rat promoter sequence from -175 to -168 is a CBF1-binding site. To test in vivo whether the rat renin CBF1-binding site functions as a binding site for CBF1 and target for activation by the Notch signaling pathway, a construct containing seven tandem copies of the rat renin CBF1-binding site inserted immediately upstream an SV40 promoter (7XrCBF1-SV40) was cotransfected into COS-7 cells with an expression vector for CBF1, VP16-CBF1, or N1IC. Results showed that overexpression of CBF1 did not have a significant effect on promoter activity, whereas VP16-CBF1 or N1IC activated the promoter by >20- or >200-fold, respectively (Fig. 1C). Moreover, neither VP16-CBF1 nor N1IC has any stimulatory effect on activity of the promoter containing five tandem copies of the mutated rat renin CBF1-binding site (5XmrCBF1-SV40). These results indicate that the rat renin CBF1-binding site is capable of binding the CBF1·N1IC complex in vivo. Results from transfection analysis also showed that N1IC-induced promoter activity of construct 7XrCBF1-SV40 was reduced by overexpression of CBF1, consistent with previous reports (17Oswald F. Liptay S. Adler G. Schmid R.M. Mol. Cell. Biol. 1998; 18: 2077-2088Crossref PubMed Google Scholar, 18Chen H. Chong Y. Liu C.-L. Biochemistry. 2000; 39: 1675-1682Crossref PubMed Scopus (12) Google Scholar). Identification of an Ets-binding Site within the Rat Renin Promoter—An Ets-binding site has been identified in the human renin promoter, which resides immediately 3′ to the TATA box (19Borensztein P. Germain S. Fuchs S. Philippe J. Corvol P. Pinet F. Circ. Res. 1994; 74: 764-773Crossref PubMed Scopus (67) Google Scholar). However, no Ets-binding site is identifiable at the same position in the rat renin promoter. The rat renin promoter sequence does, however, contain several GGA(A/T) (or inverting (T/A)TCC) motifs, which are the consensus binding sequences for the Ets family transcription factors. Oligonucleotide (rR150), which contains a putative Ets-binding site located between -143 and -138 bp of the rat renin promoter, was found to bind nuclear proteins prepared from As4.1 cells (Fig. 2). Competition EMSA using a 100-fold molar excess of unlabeled competitor oligonucleotides containing truncations and mutations within rR150 confirmed that the critical base pairs for nuclear protein binding include TTCC. Moreover, the DNA·protein complexes in EMSA are efficiently competed by an oligonucleotide (Fig. 2, conEts) containing the consensus Ets-binding site. These results demonstrate that the rat renin promoter contains an Ets-binding site located between -143 and -138 bp. Effect of Mutation in the CBF1-, Ets-, or HOX·PBX-binding Site on Promoter Activity of the Rat Renin Gene—To test whether mutation of the CBF1-, Ets-, or HOX·PBX-binding site affects activity of the rat renin promoter, constructs containing single, double, or triple mutations in these sites (Fig. 3) were transfected into Calu-6, As4.1, or COS-7 cells. Calu-6 cells were developed from a human pulmonary carcinoma and express their endogenous renin gene (20Lang J.A. Yang G. Kern J.A. Sigmund C.D. Hypertension. 1995; 25: 704-710Crossref PubMed Google Scholar), whereas COS-7 cells do not express their endogenous renin gene but have been widely used to study the Notch signaling pathway. Consistent with this, a 60-fold higher basal expression from the rat renin promoter was observed in Calu-6 cells than in COS-7 cells (Fig. 4). Moreover, mutation of the CBF1-binding site resulted in a significant increase in promoter activity in both Calu-6 and COS-7 cells, indicating that CBF1 binds to the rat renin promoter and acts as a transcriptional repressor. However, mutation of the CBF1-binding site had no effect on promoter activity in As4.1 cells, suggesting that one or more co-repressors necessary for CBF1-mediated repression may be absent in As4.1 cells. Alternatively, the Notch pathway may be constitutively active in these cells. Mutation of the Ets-binding site reduced promoter activity by 63, 33, and 78% in Calu-6, As4.1, and COS-7 cells, respectively, demonstrating an important role for Ets family transcription factors in regulating rat renin gene expression. Our results also show that mutation of the HOX·PBX-binding site alone did not significantly alter transcriptional activity of the rat renin promoter in these three cell lines. Only when the HOX·PBX-binding site is mutated in a construct that already contains mutations in the CBF1- and Ets-binding sites was a significant decrease in promoter activity observed in As4.1 cells (compare promoter activity of rR240/mEC with that of rR240/mPHEC). Another interesting finding was that mutation of the CBF1-binding site did not significantly increase transcriptional activity of the rat renin promoter lacking the Ets-binding site in any of the three cell lines (compare promoter activity of rR240/mEts with that of rR240/mEC), suggesting that the repressor function of CBF1 may be dependent on the presence of the Ets-binding site. Activation of the Rat Renin Promoter by HOX D10·PBX1b· PREP1, Ets-1, and N1IC in COS-7 Cells—COS-7 cells were cotransfected with the reporter construct rR240 and expression vectors for N1IC, Ets-1, and/or Ets-2 to determine whether these transcription factors are capable of activating the rat renin promoter individually or in combination. As shown in Fig. 5A, either N1IC or Ets-1 by itself activated the promoter by 2-fold, whereas overexpression of Ets-2 did not have any effect. The Ets-2-expressing vector used in this study could activate an Ets-responsive human chorionic gonadotropin subunit α-promoter-luciferase reporter (38Ghosh D. Sachdev S. Hannink M. Roberts R.M. Mol. Endocrinol. 2005; 19: 1049-1066Crossref PubMed Scopus (21) Google Scholar) in COS-7 cells (data not shown), indicating that active Ets-2 protein is generated from the vector. N1IC was capable of further increasing the Ets-1-mediated transactivation to 6-fold, indicating that N1IC and Ets-1 activate the rat renin promoter cooperatively. Ets-2, however, was incapable of collaborating with N1IC (data not shown). When COS-7 cells were transfected with the reporter construct rR240/mCBF1, which contains a mutation in the CBF1-binding site, induction by N1IC on basal promoter activity was reduced to 1.4-fold. Moreover, N1IC had no effect on the Ets-1-mediated activation. These results suggest that the CBF1-binding site is important for the function of N1IC. Finally, when the renin promoter construct containing the mutation in the Ets-binding site (rR240/mEts) was tested, no induction of promoter activity by either N1IC or Ets-1, or both, was observed, showing that the Ets-binding site is necessary not only for the effect of Ets-1 on the rat renin promoter but for the effect of N1IC as well. We next examined whether HOXD10·PBX1b·PREP1, which has been shown to bind to the HOX·PBX-binding site in the mouse, rat, or human renin promoter (Ref. 6Pan L. Xie Y. Black T.A. Jones C.A. Pruitt S.C. Gross K.W. J. Biol. Chem. 2001; 276: 32489-32494Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar and data not shown), is capable of activating the rat renin promoter alone or in cooperation with N1IC in COS-7 cells. Results from transfection assays showed that HOXD10 alone activated the rat renin promoter in construct rR240 by 2.3-fold, whereas either PBX1b or PREP1 alone had no effect (Fig. 5B). Moreover, cotransfection of both PBX1b and PREP1 caused a 2-fold increase in rat renin promoter activity. HOXD10 further enhances the induction by PBX1b and PREP1 to 5.3-fold. The HOX·PBX-binding site is important for the activation of the rat renin promoter by HOXD10, PBX1b, and PREP1 since mutation of the site resulted in a 2.7-fold decrease in the activation by these homeodomain proteins. However, mutation of the HOX·PBX binding site did not completely abolish the induction by HOXD10·PBX1b·PREP1, suggesting that there may be one or more cis-elements present in the rat renin promoter mediating their effect. Our results also showed a cooperative activation of the rat renin promoter by N1IC and HOXD10·PBX1b· PREP1. The addition of N1IC caused a 3.8-fold further enhancement of the induction by HOXD10·PBX1b·PREP1 of promoter activity of the rat renin gene. The enhancement by N1IC of the HOXD10·PBX1b·PREP1 induction was reduced to 1.7- or 2.5-fold when the CBF1- or HOXD10·PBX1b·PREP1-binding site, respectively, was mutated. When COS-7 cells were cotransfected with expression vectors for Ets-1, HOXD10, PBX1b, and PREP1 altogether with rRen240, a 14.4-fold induction over basal promoter activity of the rat renin gene was observed (Fig. 5C). N1IC further enhanced this induction to 31.4-fold. Interestingly, N1IC decreased Ets-1·HOXD10·PBX1b·PREP1-mediated induction by 2-fold in COS-7 cells transfected with the rat renin promoter containing the mutated CBF1-binding site. Although Ets-1 alone had no effect on activity of the rat renin promoter containing the mutated Ets-1-binding site, it is capable of further increasing the HOXD10·PBX1b·PREP1-mediated activation of the same promoter. It is possible that Ets-1 may directly or indirectly interact with the HOXD10·PBX1b·PREP1 complex. Moreover, overexpression of N1IC did not alter the effect of Ets-1·HOXD10·PBX1b·PREP1 on activity of this mutant promoter, demonstrating again that not only is the activation of the rat renin promoter by N1IC dependent on the presence of the CBF1-binding site but on the Ets-1-binding site as well. On the contrary, mutation of the HOX·PBX-binding site did not change the effect of N1IC. A 2-fold induction by N1IC over the Ets-1·HOXD10·PBX1b·PREP1-mediated activation of the promoter was still observed. Finally, when the construct rRen240/mPHEC, in which the HOX·PBX-, Ets-1-, and CBF1-binding sites are all mutated, was tested in COS-7 cells, no significant effect from N1IC, Ets-1, or HOXD10·PBX1b·PREP1 by themselves on promoter activity was detected. When compared with the 31.4-fold activation of the wild-type rat renin promoter, only a 2-fold activation of this promoter was observed in COS-7 cells simultaneously expressing Ets-1, HOXD10, PBX1b, PREP1, and N1IC, suggesting critical roles for these three cis-regulatory sites in regulating renin gene expression. In this study, a CBF1- and an Ets-binding site were identified in the promoter region of the rat renin gene. Transcription factor Ets-1 binds to the Ets-binding site and activates the rat renin promoter, whereas CBF1 acts as a transcriptional repressor in renin-expressing Calu-6 and non-renin-expressing COS-7 cells. Moreover, N1IC is capable of counteracting the negative effect of CBF1 and activating the rat renin promoter via the CBF1-binding site in cooperation with Ets-1 and HOXD10·PBX1b·PREP1. The Ets transcription factors are implicated in cellular proliferation, differentiation, migration, apoptosis, and cell-cell interactions (21Maroulakou I.G. Bowe D.B. Oncogene. 2000; 19: 6432-6442Crossref PubMed Scopus (157) Google Scholar). An Ets-binding site has also been identified in the human renin promoter (19Borensztein P. Germain S. Fuchs S. Philippe J. Corvol P. Pinet F. Circ. Res. 1994; 74: 764-773Crossref PubMed Scopus (67) Google Scholar). It is located immediately 3′ to the TATA box and capable of binding Ets-1 (22Konoshita T. Germain S. Philippe J. Corvol P. Pinet F. Kidney Int. 1996; 50: 1515-1524Abstract Full Text PDF PubMed Scopus (18) Google Scholar, 23Konoshita T. Makino Y. Wakahara S. Ido K. Yoshida M. Kawai Y. Miyamori I. J. Cell. Biochem. 2004; 93: 327-336Crossref PubMed Scopus (13) Google Scholar). However, whether it contributes to promoter activity of the human renin gene has not been determined. Ets-1 has been reported to play a role in vascular development and angiogenesis (24Sato Y. Cell Struct. Funct. 2001; 26: 19-24Crossref PubMed Scopus (111) Google Scholar). Mice deficient for Ets-1 have severe kidney abnormalities and/or lethal angiogenic defects (25Gomez R.A. J. Histochem. Cytochem. 1999; 47: 1643PubMed Google Scholar). Considering that renin-expressing cells are associated with the branching of renal arterioles (26Reddi V. Zaglul A. Pentz E.S. Gomez R.A. J. Am. Soc. Nephrol. 1998; 9: 63-71Crossref PubMed Google Scholar, 27Jones C.A. Hurley M.I. Black T.A. Kane C.M. Pan L. Pruitt S.C. Gross K.W. Physiol. Genomics. 2000; 4: 75-81Crossref PubMed Scopus (48) Google Scholar), Ets-1 may be one of transcription factors critical for renin gene expression. Whether any of the Ets consensus sequences present in the mouse renin promoter is a functional Ets-binding site remains to be investigated. The Notch signaling pathway also plays an important role in forming the vasculature (28Shawber C.J. Kitajewski J. BioEssays. 2004; 26: 225-234Crossref PubMed Scopus (195) Google Scholar). Mice deficient in genes encoding Notch, Notch ligands, and components of the Notch signaling pathway all show vascular defects. Notch and Notch ligands are expressed throughout the vasculature early in embryonic development but restricted to arterial vessels later. This is consistent with the notion that renin expression is only detected in developing arteries in the mouse or rat fetal kidney (29Jones C.A. Sigmund C.D. McGowan R.A. Kane-Haas C.M. Gross K.W. Mol. Endocrinol. 1990; 4: 375-383Crossref PubMed Scopus (102) Google Scholar, 30Gomez R.A. Lynch K.R. Sturgill B.C. Elwood J.P. Chevalier R.L. Carey R.M. Peach M.J. Am. J. Physiol. 1989; 257: F850-F858PubMed Google Scholar). Identification of the renin gene as the downstream target of the Notch signaling pathway provides us with some clues in understanding the tissue-specific regulation of renin gene expression. We have previously hypothesized that the binding of transcriptional repressors to the renin promoter may be the reason that renin transcription is turned off in non-renin-expressing cells (2Petrovic N. Black T.A. Fabian J.R. Kane C. Jones C.A. Loudon J.A. Abonia J.P. Sigmund C.D. Gross K.W. J. Biol. Chem. 1996; 271: 22499-22505Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Here, we show that CBF1 is one of those hypothesized repressors. Upon activation of notch signaling, the direct interaction of NIC with CBF1 turns CBF1 from a repressor to an activator. However, the renin gene is not expressed in every cell in which Notch signaling is activated, suggesting that Notch is necessary but not sufficient for renin gene expression. Cooperation between CBF1·Notch and other transcription factors such as Ets-1 and HOX D10· PBX1b·PREP1 may be necessary for renin expression. This fits a model proposed by Furriols and Bray (31Furriols M. Bray S. Curr. Biol. 2001; 11: 60-64Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar) for Notch target gene regulation. They suggest that in the absence of Notch, DNA-bound CBF1 prevents transcriptional activators from enhancing transcription. Notch activation can alleviate the repression so that CBF1 is capable of cooperating with other DNA-bound activators to promote transcription. We observed that N1IC had reduced but still significant stimulatory effect on the promoter activity of the rat renin gene even when the CBF1-binding site is mutated. However, when the Ets-binding site is mutated, no significant effect by N1IC is observed. It is possible that N1IC directly or indirectly interacts with DNA-bound Ets-1 to promote this increase. Interactions of N1IC with transcription factors other than CBF1 have been reported. For example, N1IC is capable of inhibiting NF-κB activity in the nucleus by a direct interaction (32Wang J. Shelly L. Miele L. Boykins R. Norcross M.A. Guan E. J. Immunol. 2001; 167: 289-295Crossref PubMed Scopus (108) Google Scholar). Moreover, N1IC can act as a co-activator for transcription factor LEF-1 (33Ross D.A. Kadesch T. Mol. Cell. Biol. 2001; 21: 7537-7544Crossref PubMed Scopus (90) Google Scholar). We showed that the wild-type rat renin promoter or the promoter containing triple mutations in the HOX·PBX-, Ets-, and CBF1-binding site exhibits considerably stronger activity in renin-expressing Calu-6 than in COS-7 cells (Fig. 4). This suggests that there are still unidentified cell-specific transcription factors binding at the rat renin promoter important for expression and that the Calu-6 cell line can be used as a model system to identify these transcription factors. These unidentified transcription factors may also cooperate with Notch to promote renin transcription. A number of transcription factor-binding sites have been identified in the human renin promoter using primary cultures of renin-expressing human chorionic cells (19Borensztein P. Germain S. Fuchs S. Philippe J. Corvol P. Pinet F. Circ. Res. 1994; 74: 764-773Crossref PubMed Scopus (67) Google Scholar). In addition to the HOX·PBX- and Ets-binding site, a cAMP-responsive element, a chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII)-binding site, and two unidentified transcription factor-binding sites are present in the human promoter. These cis-regulatory sites could also be present in the rat renin promoter considering that strong sequence conservation is found in this region upon aligning these two promoters (1Pan L. Gross K.W. Hypertension. 2005; 45: 3-8Crossref PubMed Scopus (73) Google Scholar). The rat renin promoter with the mutation in the CBF1-binding site did not show any increased activity when compared with the wild-type promoter when assayed in As4.1 cells. This result suggests that the repressive function of CBF1 is lost in As4.1 cells. However, results from EMSA show that CBF1 is expressed in As4.1 cells (Fig. 1B). It is possible that one or more co-repressors are absent or that the Notch pathway is constitutively active in this cell line. Since As4.1 cells were selected from tumors initiated by transgene-targeted oncogenesis with a mouse renin promoter driving SV40 T antigen, the renin gene promoter is probably maximally stimulated, and any negative regulation of the renin promoter may be lost or diminished in these cells. We showed previously that high level expression of Ren-1c in As4.1 cells is partly due to a constitutively active cAMP-dependent protein kinase (3Pan L. Black T.A. Shi Q. Jones C.A. Petrovic N. Loudon J. Kane C. Sigmund C.D. Gross K.W. J. Biol. Chem. 2001; 276: 45530-45538Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). It has been reported that the intracellular domain of Notch3 (N3IC), in contrast to N1IC, is a poor activator and is capable of acting as a repressor by blocking the N1IC-activated gene expression (34Beatus P. Lundkvist J. Oberg C. Lendahl U. Development (Camb.). 1999; 126: 3925-3935Crossref PubMed Google Scholar). However, we have found that N3IC can also cooperate with Ets-1 or HOXD10·PBX1b·PREP1 to activate the rat renin promoter, although the effect by N3IC is smaller than that of N1IC (data not shown). Moreover, an additive effect was obtained when both N1IC and N3IC were cotransfected with the promoter-reporter construct in the presence or absence of other expression vectors (data not shown). These results suggest that members of the Notch family may have redundant roles in regulating renin gene expression. We are currently studying the renin expression pattern in mice deficient for Notch3 expression (35Krebs L.T. Xue Y. Norton C.R. Sundberg J.P. Beatus P. Lendahl U. Joutel A. Gridley T. Genesis. 2003; 37: 139-143Crossref PubMed Scopus (198) Google Scholar), which have been shown to have defects in arterial differentiation and maturation of vascular smooth muscle cells (36Domenga V. Fardoux P. Lacombe P. Monet M. Maciazek J. Krebs L.T. Klonjkowski B. Berrou E. Mericskay M. Li Z. Tournier-Lasserve E. Gridley T. Joutel A. Genes Dev. 2004; 18: 2730-2735Crossref PubMed Scopus (378) Google Scholar), to further understand the mechanisms of regulation of renin gene expression by Notch. A screen of the Drosophila X chromosome for genes whose dosage affects the function of the HOX gene Deformed revealed that Notch is one of these genes (37Florence B. McGinnis W. Genetics. 1998; 150: 1497-1511Crossref PubMed Google Scholar). Notch also affects the function of another HOX gene, Ultrabithorax. These results suggest that Notch may be generally involved in homeotic function. Here, we show that Notch signaling and HOX·PBX· PREP complex functionally interact with each other to activate renin gene expression. The finding suggests a role for renin in the development of renal vasculature. We thank Dr. R. Kopan for providing the mouse N1IC expression vector. We also thank Colleen Kane for cell culture preparation. This research utilized core facilities supported in part by Roswell Park Cancer Institute's National Cancer Institute-funded Cancer Center support Grant CA-16056.