- Email: [email protected]
Characterization of the murine endothelial nitric oxide synthase promoter
Biochimica et Biophysica Acta 1443 (1998) 352^357
Promoter paper
Characterization of the murine endothelial nitric oxide synthase promoter Anouk-Martine Teichert, Fotula Karantzoulis-Fegaras, Yang Wang, Imtiaz A. Mawji, Xei Bei, Kumudini Gnanapandithen, Philip A. Marsden * Renal Division and Department of Medicine, St. Michael's Hospital and University of Toronto, 1 Kings College Circle, Toronto, Ont. M5S 1A8, Canada Received 16 September 1998; accepted 23 October 1998
Abstract As our understanding of the contributory roles of NO in the blood vessel wall evolves, so does the need to firmly understand the basic principles governing the regulated expression of the endothelial nitric oxide synthase (eNOS) gene. Because a robust approach to dissecting the relative contribution of a given cardiovascular gene exploits the use of murine genetic models, P1 murine genomic clones were isolated, characterized and functionally assessed to gain further insight into the regulated expression of the eNOS gene in the mouse. Sequence analysis of 1.8 kb of 5P flanking regions revealed important regions of sequence conservation with human and bovine sequences. Functional promoter activity was confirmed using transient transfection analysis of cultured endothelial cells. ß 1998 Elsevier Science B.V. All rights reserved. Keywords: Atherosclerosis ; Endothelium; Hypertension; Nitric oxide; Gene regulation; Promoter
Local release and action of nitric oxide (NO) in blood vessels represents a potent and e¡ective regulator of vascular tone and organ blood £ow. Endothelial nitric oxide synthase (eNOS) is the enzyme responsible, in major part, for endothelial-derived NO [1,2]. Targeted inactivation of the murine eNOS locus by homologous recombination and physiologic assessment of (3/3) o¡spring has reinforced the viewpoint that NO in blood vessels plays a quintessential role in regulation of local blood vessel tonus [3,4], remodelling of the vascular wall in response to changes in £ow or distending hydrostatic Abbreviations: ORF, open reading frame; UTR, untranslated region * Corresponding author. Fax: +1 (416) 978-8765; E-mail: [email protected]
pressure and modulation of hemostatic pathways [5]. In situ cRNA hybridization studies performed in a wide variety of human tissues revealed that eNOS mRNA transcripts are relatively endothelial cell-speci¢c [6,7]. This contrasts with the broad tissue distribution of the other known members of the human NOS gene family, namely neuronal NOS and inducible NOS [8^10]. Regulation of eNOS at the biochemical and enzymatic level is now better understood. eNOS is a peripheral membrane protein that is localized to specialized cell surface microdomains implicated in signal transduction known as plasmalemmal caveolae [11]. N-Myristoylation and palmitoylation are necessary for e¤cient targeting and membrane insertion [12]. Increases in intracellular calcium following endothelial activation facilitate interactions between the
0167-4781 / 98 / $ ^ see front matter ß 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 9 8 ) 0 0 2 3 6 - X
BBAEXP 91215 10-12-98
A.-M. Teichert et al. / Biochimica et Biophysica Acta 1443 (1998) 352^357
353
Fig. 1. Nucleotide sequence of the murine eNOS promoter (1.8 kb), included is the ¢rst exon and 66 nt of the ¢rst intron. Putative transfactor binding sites of the coding and non-coding strands are illustrated. Numbering is with respect to transcription initiation. Sequences depicted in this ¢gure have been deposited in GenBank (accession number: AF091262).
BBAEXP 91215 10-12-98
354 A.-M. Teichert et al. / Biochimica et Biophysica Acta 1443 (1998) 352^357
BBAEXP 91215 10-12-98 Fig. 2. Cross-species alignment of the murine, bovine and human eNOS proximal promoter regions. Enclosed sequences denote regions of nucleotide identity. Conserved transfactor binding sites are illustrated.
A.-M. Teichert et al. / Biochimica et Biophysica Acta 1443 (1998) 352^357
355
Table 1 Functional activity of the murine eNOS 5P £anking region Construct
Relative L-galactosidase activitya (1034 units/mg) þ S.E.M.
Pnls LacZ P-5200/+28 Mu eNOSnls LacZ
0.48 þ 0.17 5.85 þ 0.60
a
Fold activity 1 12.3
L-Galactosidase activity is normalized for mock transfected background.
eNOS apoenzyme and calmodulin. This enhances NADPH-dependent electron £ux through eNOS dimers resulting in the 5 electron oxidation of L-arginine and release of NO. Surprisingly, the molecular chaperone Hsp90 is also recruited to eNOS following cellular activation [13]. Recent studies have highlighted the important contributions of changes in steady-state eNOS mRNA transcripts to the regulated expression of NO in disease states in vivo and in models of endothelial activation in vitro. For instance, impairment in the bioactivity of endothelial-derived NO may be mediated, in part, through decreased expression of the mRNA transcript and protein for eNOS in atherosclerotic human blood vessels [7]. Lysophosphatidylcholine [14], shear stress [15], estrogen [16] and TGFL [17] represent important examples of exogenous stimuli known to modify eNOS gene transcription. An intriguing facet of the control of steady-state eNOS mRNA expression in vascular endothelium is the contribution of post-transcriptional regulation [6,18^21]. The regulation eNOS mRNA stability in response to exogenous stimuli, especially the mechanism by which the transcript is degraded, is an evolving story. Recently, this laboratory and others reported the isolation and characterization of complementary and genomic clones for eNOS [6,22,23]. The eNOS gene is present as a single copy in the haploid genome and has been localized to 7q35^36 in man [23^25] and chromosome 5 in the mouse [26]. Structural characterization of human genomic DNA revealed that the 4052 nt mRNA is derived from 26 exons distributed over 21 kb of human genomic DNA [23^25]. Even though analysis of 5P £anking regions failed to de¢ne a canonical TATAA motif, a single major transcription initiation site was de¢ned by primer extension, S1 nuclease protection and 5P-RACE to be 22 nt upstream of the translational start site [23]. Studies suggested a prominent role for Sp1 and GATA cis-
elements in constitutive transcription initiation [23^ 25,27,28]. This report describes the isolation and sequence analysis of 1.8 kb of the murine eNOS 5P £anking region. The nt and deduced amino acid sequence of the murine eNOS cDNA has been characterized [29] and revealed a ORF of 3606 bp, corresponding to a predicted protein of 1202 amino acids with a molecular weight of 132 kDa. Murine eNOS exhibited 94 and 93% amino acid identity with human and bovine sequences, respectively. Murine genomic clones for eNOS were isolated from a P1 bacteriophage murine diploid Mus musculus cv129 strain genomic library (Genome Systems, St. Louis, MO) using a PCRbased screening method. A primer pair generating a 91 bp amplicon speci¢c for exon 1 of murine eNOS (sense: 5P-GCATGGGCAACTTGAAGAGT3P; antisense: 5P-CCCTGCTTGCCGCACAGC-3P) was designed using Oligo 5.0 (National Biosciences, Plymouth, MN). PCR conditions included 25 pmol of each primer, 1.5 mM MgCl2 , 200 WM dNTPs and 1.25 U Taq polymerase (Gibco/Life Technologies, Gaithersburg, MD) in ¢nal reaction volumes of 50 Wl. Cycling parameters included an initial denaturation at 94³C for 5 min, then 30 cycles at 94³C for 30 s, 56.6³C for 30 s and 72³C for 30 s, followed by a ¢nal extension of 72³C for 15 min. Restriction enzyme mapping and DNA sequence analysis of three independent clones con¢rmed authenticity. Intronic sequence was detected con¢rming that they did not represent processed pseudogenes. P1 clones were partially digested with Sau3A into 5^10 kb fragments and subcloned into pBluescript SK(3) (Stratagene, La Jolla, CA), as previously described [30]. A plasmid clone containing the murine 5P £anking regions and initial exons was identi¢ed. 1.8 kb of the murine 5P £anking regions was subjected to sequence analysis on both strands and is shown in Fig. 1. As with the human promoter, murine eNOS is a TATA-less promoter with multiple Sp1, GATA and Ets member
BBAEXP 91215 10-12-98
356
A.-M. Teichert et al. / Biochimica et Biophysica Acta 1443 (1998) 352^357
cis-regulatory elements. A sequence comparison of the murine, human and bovine proximal promoter regions was performed and is shown in Fig. 2. A high degree of sequence identity is evident, though 5P-UTR sequences were not conserved. Sequences reported in this paper have been deposited in GenBank (accession number: AF091262). To de¢ne whether these murine genomic sequences exhibit functional promoter activity, a promoter/reporter L-galactosidase reporter construct was created (p-5200/+28 Mu eNOSnls LacZ) comprising 5.2 kb of murine eNOS 5P £anking regions (35.2 kb to +28 nt) directing nuclear localized L-galactosidase (pnls LacZ) [31]. 3P-UTR and 3P £anking genomic sequences were derived from the human K1 -globin gene. Transient transfections were carried out using the Lipofectin Reagent (Gibco BRL). Bovine aortic endothelial cells were cultured as previously described [20,32] and plated at 3.3U104 cells/ml (3.5 ml) on 60 mm dishes 48 h prior to transfection. Cells were co-transfected with 1.0 Wg of promoter/reporter construct, 0.5 Wg of pGL3 Control (SV40 promoter and enhancer directing luciferase reporter) and 1.5 Wg of pBluescript II SK(3) DNA. pRSV-L-gal was used as a representative strong heterologous promoter and pnis LacZ was used as a promoterless construct for positive and negative controls, respectively. Luciferase activity was assessed to control for transfection e¤ciency and pBluescript II SK(3) DNA was used to optimize DNA/Lipofectin ratios and hence transfection e¤ciency. DNA/Lipofectin in a 1:2 (mass:mass) ratio was incubated for 60 min at 22³C then added to cells at 37³C in serum-free OPTI-MEM I. The transfection mix was replaced at 5 h with RPMI 1640 supplemented with 15% bovine calf serum. Each transfection experiment was performed in triplicate in three independent endothelial cell clones. Results shown in Table 1 represent the mean of three independent experiments (triplicate determinations) and con¢rm functional promoter activity of these murine genomic regions. The cloning and characterization of murine eNOS promoter regions o¡ers the opportunity for future studies de¢ning transcriptional regulation of the murine eNOS gene at the molecular level and transgenic in vivo approaches. P.A.M. is suppported by a Grant from the Medical Research Council of Canada (GR-13298) and a
Career Investigator Award from the Heart and Stroke Foundation of Canada.
References [1] T. Michel, O. Feron, J. Clin. Invest. 100 (1997) 2146^ 2152. [2] D.G. Harrison, J. Clin. Invest. 100 (1997) 2153^2157. [3] P.L. Huang, Z. Huang, H. Mashimo, K.D. Bloch, M.A. Moskowitz, J.A. Bevan, M.C. Fishman, Nature 377 (1995) 239^242. [4] E.G. Shesely, N. Maeda, H.S. Kim, K.M. Desai, J.H. Krege, V.E. Laubach, P.A. Sherman, W.C. Sessa, O. Smithies, Proc. Natl. Acad. Sci. USA 93 (1996) 13176^13181. [5] R.D. Rudic, E.G. Shesely, N. Maeda, O. Smithies, S.S. Segal, W.C. Sessa, J. Clin. Invest. 101 (1998) 731^736. [6] P.A. Marsden, K.T. Shappert, H.S. Chen, M. Flowers, C.L. Sundell, J.N. Wilcox, S. Lamas, T. Michel, FEBS Lett. 307 (1992) 287^293. [7] J.N. Wilcox, R.R. Subramanian, C.L. Sundell, W.R. Tracey, J.S. Pollock, D.G. Harrison, P.A. Marsden, Arterioscler. Thromb. Vasc. Biol. 17 (1997) 2479^2488. [8] Y. Wang, P.A. Marsden, Curr. Opin. Nephrol. Hypertens. 4 (1995) 12^22. [9] T.M. Dawson, V.L. Dawson, S.H. Snyder, Ann. Neurol. 32 (1992) 297^311. [10] C.J. Lowenstein, J.L. Dinerman, S.H. Snyder, Ann. Intern. Med. 120 (1994) 227^237. [11] P. Shaul, E. Smart, L. Robinson, Z. German, I. Yuhanna, Y. Ying, R. Anderson, T. Michel, J. Biol. Chem. 271 (1996) 6518^6522. [12] L. Belhassen, O. Feron, D.M. Kaye, T. Michel, R.A. Kelly, J. Biol. Chem. 272 (1997) 11198^11204. [13] G. Garcia-Cardena, R. Fan, V. Shah, R. Sorrentino, G. Cirino, A. Papapetropoulos, W.C. Sessa, Nature 392 (1998) 821^824. [14] K. Cieslik, A. Zembowicz, J.L. Tang, K.K. Wu, J. Biol. Chem. 273 (1998) 14885^14890. [15] M. Uematsu, Y. Ohara, J.P. Navas, K. Nishida, T.J. Murphy, R.W. Alexander, R.M. Nerem, D.G. Harrison, Am. J. Physiol. 269 (1995) C1371^C1378. [16] H. Kleinert, T. Wallerath, C. Euchenhofer, I. Ihrig-Biedert, H. Li, U. Forstermann, Hypertension 31 (1998) 582^588. [17] N. Inoue, R.C. Venema, H.S. Sayegh, Y. Ohara, T.J. Murphy, D.G. Harrison, Arterioscler. Thromb. Vasc. Biol. 15 (1995) 1255^1261. [18] M. Yoshizumi, M. Perrella, J.J. Burnett, M. Lee, Circ. Res. 73 (1993) 205^209. [19] L.P. McQuillan, G.K. Leung, P.A. Marsden, S.K. Kostyk, S. Kourembanas, Am. J. Physiol. 267 (1994) H1921^H1927. [20] M.A. Flowers, Y. Wang, R. Stewart, B. Patel, P.A. Marsden, Am. J. Physiol. 269 (1995) H1988^H1997. [21] J. Liao, W. Shin, W. Lee, S. Clark, J. Biol. Chem. 270 (1995) 319^324.
BBAEXP 91215 10-12-98
A.-M. Teichert et al. / Biochimica et Biophysica Acta 1443 (1998) 352^357 [22] T. Collins, M. Read, A. Neish, M. Whitley, D. Thanos, T. Maniatis, FASEB J. 9 (1995) 899^909. [23] P.A. Marsden, H.H. Heng, S.W. Scherer, R.J. Stewart, A.V. Hall, X.M. Shi, L.C. Tsui, K.T. Schappert, J. Biol. Chem. 268 (1993) 17478^17488. [24] S. Nadaud, A. Bonnardeaux, M. Lathrop, F. Soubrier, Biochem. Biophys. Res. Commun. 198 (1994) 1027^1033. [25] L. Robinson, S. Weremowicz, C. Morton, T. Michel, Genomics 19 (1994) 350^357. [26] A.R. Gregg, C.G. Lee, G.E. Herman, W.E. O'Brien, Mamm. Genome 6 (1995) 152. [27] S. Wariishi, K. Miyahara, K. Toda, S. Ogoshi, Y. Doi, S. Ohnishi, Y. Mitsui, Y. Yui, C. Kawai, Y. Shizuta, Biochem. Biophys. Res. Commun. 216 (1995) 729^735.
357
[28] R. Zhang, W. Min, W.C. Sessa, J. Biol. Chem. 270 (1995) 15320^15326. [29] K. Gnanapandithen, Z. Chen, C.-L. Kau, R.M. Gorczynski, P.A. Marsden, Biochim. Biophys. Acta 1308 (1996) 103^ 106. [30] A.V. Hall, H. Antoniou, Y. Wang, A. Cheung, A. Arbus, S. Olson, W.C. Liu, C.L. Kau, P.A. Marsden, J. Biol. Chem. 269 (1994) 33082^33090. [31] J.J. Peschon, R.R. Behringer, R.L. Brinster, R.D. Palmiter, Proc. Natl. Acad. Sci. USA 84 (1987) 5316^5319. [32] P.A. Marsden, T. Brock, B. Ballermann, Am. J. Physiol. 258 (1990) F1295^F1303.
BBAEXP 91215 10-12-98
Promoter paper
Characterization of the murine endothelial nitric oxide synthase promoter Anouk-Martine Teichert, Fotula Karantzoulis-Fegaras, Yang Wang, Imtiaz A. Mawji, Xei Bei, Kumudini Gnanapandithen, Philip A. Marsden * Renal Division and Department of Medicine, St. Michael's Hospital and University of Toronto, 1 Kings College Circle, Toronto, Ont. M5S 1A8, Canada Received 16 September 1998; accepted 23 October 1998
Abstract As our understanding of the contributory roles of NO in the blood vessel wall evolves, so does the need to firmly understand the basic principles governing the regulated expression of the endothelial nitric oxide synthase (eNOS) gene. Because a robust approach to dissecting the relative contribution of a given cardiovascular gene exploits the use of murine genetic models, P1 murine genomic clones were isolated, characterized and functionally assessed to gain further insight into the regulated expression of the eNOS gene in the mouse. Sequence analysis of 1.8 kb of 5P flanking regions revealed important regions of sequence conservation with human and bovine sequences. Functional promoter activity was confirmed using transient transfection analysis of cultured endothelial cells. ß 1998 Elsevier Science B.V. All rights reserved. Keywords: Atherosclerosis ; Endothelium; Hypertension; Nitric oxide; Gene regulation; Promoter
Local release and action of nitric oxide (NO) in blood vessels represents a potent and e¡ective regulator of vascular tone and organ blood £ow. Endothelial nitric oxide synthase (eNOS) is the enzyme responsible, in major part, for endothelial-derived NO [1,2]. Targeted inactivation of the murine eNOS locus by homologous recombination and physiologic assessment of (3/3) o¡spring has reinforced the viewpoint that NO in blood vessels plays a quintessential role in regulation of local blood vessel tonus [3,4], remodelling of the vascular wall in response to changes in £ow or distending hydrostatic Abbreviations: ORF, open reading frame; UTR, untranslated region * Corresponding author. Fax: +1 (416) 978-8765; E-mail: [email protected]
pressure and modulation of hemostatic pathways [5]. In situ cRNA hybridization studies performed in a wide variety of human tissues revealed that eNOS mRNA transcripts are relatively endothelial cell-speci¢c [6,7]. This contrasts with the broad tissue distribution of the other known members of the human NOS gene family, namely neuronal NOS and inducible NOS [8^10]. Regulation of eNOS at the biochemical and enzymatic level is now better understood. eNOS is a peripheral membrane protein that is localized to specialized cell surface microdomains implicated in signal transduction known as plasmalemmal caveolae [11]. N-Myristoylation and palmitoylation are necessary for e¤cient targeting and membrane insertion [12]. Increases in intracellular calcium following endothelial activation facilitate interactions between the
0167-4781 / 98 / $ ^ see front matter ß 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 9 8 ) 0 0 2 3 6 - X
BBAEXP 91215 10-12-98
A.-M. Teichert et al. / Biochimica et Biophysica Acta 1443 (1998) 352^357
353
Fig. 1. Nucleotide sequence of the murine eNOS promoter (1.8 kb), included is the ¢rst exon and 66 nt of the ¢rst intron. Putative transfactor binding sites of the coding and non-coding strands are illustrated. Numbering is with respect to transcription initiation. Sequences depicted in this ¢gure have been deposited in GenBank (accession number: AF091262).
BBAEXP 91215 10-12-98
354 A.-M. Teichert et al. / Biochimica et Biophysica Acta 1443 (1998) 352^357
BBAEXP 91215 10-12-98 Fig. 2. Cross-species alignment of the murine, bovine and human eNOS proximal promoter regions. Enclosed sequences denote regions of nucleotide identity. Conserved transfactor binding sites are illustrated.
A.-M. Teichert et al. / Biochimica et Biophysica Acta 1443 (1998) 352^357
355
Table 1 Functional activity of the murine eNOS 5P £anking region Construct
Relative L-galactosidase activitya (1034 units/mg) þ S.E.M.
Pnls LacZ P-5200/+28 Mu eNOSnls LacZ
0.48 þ 0.17 5.85 þ 0.60
a
Fold activity 1 12.3
L-Galactosidase activity is normalized for mock transfected background.
eNOS apoenzyme and calmodulin. This enhances NADPH-dependent electron £ux through eNOS dimers resulting in the 5 electron oxidation of L-arginine and release of NO. Surprisingly, the molecular chaperone Hsp90 is also recruited to eNOS following cellular activation [13]. Recent studies have highlighted the important contributions of changes in steady-state eNOS mRNA transcripts to the regulated expression of NO in disease states in vivo and in models of endothelial activation in vitro. For instance, impairment in the bioactivity of endothelial-derived NO may be mediated, in part, through decreased expression of the mRNA transcript and protein for eNOS in atherosclerotic human blood vessels [7]. Lysophosphatidylcholine [14], shear stress [15], estrogen [16] and TGFL [17] represent important examples of exogenous stimuli known to modify eNOS gene transcription. An intriguing facet of the control of steady-state eNOS mRNA expression in vascular endothelium is the contribution of post-transcriptional regulation [6,18^21]. The regulation eNOS mRNA stability in response to exogenous stimuli, especially the mechanism by which the transcript is degraded, is an evolving story. Recently, this laboratory and others reported the isolation and characterization of complementary and genomic clones for eNOS [6,22,23]. The eNOS gene is present as a single copy in the haploid genome and has been localized to 7q35^36 in man [23^25] and chromosome 5 in the mouse [26]. Structural characterization of human genomic DNA revealed that the 4052 nt mRNA is derived from 26 exons distributed over 21 kb of human genomic DNA [23^25]. Even though analysis of 5P £anking regions failed to de¢ne a canonical TATAA motif, a single major transcription initiation site was de¢ned by primer extension, S1 nuclease protection and 5P-RACE to be 22 nt upstream of the translational start site [23]. Studies suggested a prominent role for Sp1 and GATA cis-
elements in constitutive transcription initiation [23^ 25,27,28]. This report describes the isolation and sequence analysis of 1.8 kb of the murine eNOS 5P £anking region. The nt and deduced amino acid sequence of the murine eNOS cDNA has been characterized [29] and revealed a ORF of 3606 bp, corresponding to a predicted protein of 1202 amino acids with a molecular weight of 132 kDa. Murine eNOS exhibited 94 and 93% amino acid identity with human and bovine sequences, respectively. Murine genomic clones for eNOS were isolated from a P1 bacteriophage murine diploid Mus musculus cv129 strain genomic library (Genome Systems, St. Louis, MO) using a PCRbased screening method. A primer pair generating a 91 bp amplicon speci¢c for exon 1 of murine eNOS (sense: 5P-GCATGGGCAACTTGAAGAGT3P; antisense: 5P-CCCTGCTTGCCGCACAGC-3P) was designed using Oligo 5.0 (National Biosciences, Plymouth, MN). PCR conditions included 25 pmol of each primer, 1.5 mM MgCl2 , 200 WM dNTPs and 1.25 U Taq polymerase (Gibco/Life Technologies, Gaithersburg, MD) in ¢nal reaction volumes of 50 Wl. Cycling parameters included an initial denaturation at 94³C for 5 min, then 30 cycles at 94³C for 30 s, 56.6³C for 30 s and 72³C for 30 s, followed by a ¢nal extension of 72³C for 15 min. Restriction enzyme mapping and DNA sequence analysis of three independent clones con¢rmed authenticity. Intronic sequence was detected con¢rming that they did not represent processed pseudogenes. P1 clones were partially digested with Sau3A into 5^10 kb fragments and subcloned into pBluescript SK(3) (Stratagene, La Jolla, CA), as previously described [30]. A plasmid clone containing the murine 5P £anking regions and initial exons was identi¢ed. 1.8 kb of the murine 5P £anking regions was subjected to sequence analysis on both strands and is shown in Fig. 1. As with the human promoter, murine eNOS is a TATA-less promoter with multiple Sp1, GATA and Ets member
BBAEXP 91215 10-12-98
356
A.-M. Teichert et al. / Biochimica et Biophysica Acta 1443 (1998) 352^357
cis-regulatory elements. A sequence comparison of the murine, human and bovine proximal promoter regions was performed and is shown in Fig. 2. A high degree of sequence identity is evident, though 5P-UTR sequences were not conserved. Sequences reported in this paper have been deposited in GenBank (accession number: AF091262). To de¢ne whether these murine genomic sequences exhibit functional promoter activity, a promoter/reporter L-galactosidase reporter construct was created (p-5200/+28 Mu eNOSnls LacZ) comprising 5.2 kb of murine eNOS 5P £anking regions (35.2 kb to +28 nt) directing nuclear localized L-galactosidase (pnls LacZ) [31]. 3P-UTR and 3P £anking genomic sequences were derived from the human K1 -globin gene. Transient transfections were carried out using the Lipofectin Reagent (Gibco BRL). Bovine aortic endothelial cells were cultured as previously described [20,32] and plated at 3.3U104 cells/ml (3.5 ml) on 60 mm dishes 48 h prior to transfection. Cells were co-transfected with 1.0 Wg of promoter/reporter construct, 0.5 Wg of pGL3 Control (SV40 promoter and enhancer directing luciferase reporter) and 1.5 Wg of pBluescript II SK(3) DNA. pRSV-L-gal was used as a representative strong heterologous promoter and pnis LacZ was used as a promoterless construct for positive and negative controls, respectively. Luciferase activity was assessed to control for transfection e¤ciency and pBluescript II SK(3) DNA was used to optimize DNA/Lipofectin ratios and hence transfection e¤ciency. DNA/Lipofectin in a 1:2 (mass:mass) ratio was incubated for 60 min at 22³C then added to cells at 37³C in serum-free OPTI-MEM I. The transfection mix was replaced at 5 h with RPMI 1640 supplemented with 15% bovine calf serum. Each transfection experiment was performed in triplicate in three independent endothelial cell clones. Results shown in Table 1 represent the mean of three independent experiments (triplicate determinations) and con¢rm functional promoter activity of these murine genomic regions. The cloning and characterization of murine eNOS promoter regions o¡ers the opportunity for future studies de¢ning transcriptional regulation of the murine eNOS gene at the molecular level and transgenic in vivo approaches. P.A.M. is suppported by a Grant from the Medical Research Council of Canada (GR-13298) and a
Career Investigator Award from the Heart and Stroke Foundation of Canada.
References [1] T. Michel, O. Feron, J. Clin. Invest. 100 (1997) 2146^ 2152. [2] D.G. Harrison, J. Clin. Invest. 100 (1997) 2153^2157. [3] P.L. Huang, Z. Huang, H. Mashimo, K.D. Bloch, M.A. Moskowitz, J.A. Bevan, M.C. Fishman, Nature 377 (1995) 239^242. [4] E.G. Shesely, N. Maeda, H.S. Kim, K.M. Desai, J.H. Krege, V.E. Laubach, P.A. Sherman, W.C. Sessa, O. Smithies, Proc. Natl. Acad. Sci. USA 93 (1996) 13176^13181. [5] R.D. Rudic, E.G. Shesely, N. Maeda, O. Smithies, S.S. Segal, W.C. Sessa, J. Clin. Invest. 101 (1998) 731^736. [6] P.A. Marsden, K.T. Shappert, H.S. Chen, M. Flowers, C.L. Sundell, J.N. Wilcox, S. Lamas, T. Michel, FEBS Lett. 307 (1992) 287^293. [7] J.N. Wilcox, R.R. Subramanian, C.L. Sundell, W.R. Tracey, J.S. Pollock, D.G. Harrison, P.A. Marsden, Arterioscler. Thromb. Vasc. Biol. 17 (1997) 2479^2488. [8] Y. Wang, P.A. Marsden, Curr. Opin. Nephrol. Hypertens. 4 (1995) 12^22. [9] T.M. Dawson, V.L. Dawson, S.H. Snyder, Ann. Neurol. 32 (1992) 297^311. [10] C.J. Lowenstein, J.L. Dinerman, S.H. Snyder, Ann. Intern. Med. 120 (1994) 227^237. [11] P. Shaul, E. Smart, L. Robinson, Z. German, I. Yuhanna, Y. Ying, R. Anderson, T. Michel, J. Biol. Chem. 271 (1996) 6518^6522. [12] L. Belhassen, O. Feron, D.M. Kaye, T. Michel, R.A. Kelly, J. Biol. Chem. 272 (1997) 11198^11204. [13] G. Garcia-Cardena, R. Fan, V. Shah, R. Sorrentino, G. Cirino, A. Papapetropoulos, W.C. Sessa, Nature 392 (1998) 821^824. [14] K. Cieslik, A. Zembowicz, J.L. Tang, K.K. Wu, J. Biol. Chem. 273 (1998) 14885^14890. [15] M. Uematsu, Y. Ohara, J.P. Navas, K. Nishida, T.J. Murphy, R.W. Alexander, R.M. Nerem, D.G. Harrison, Am. J. Physiol. 269 (1995) C1371^C1378. [16] H. Kleinert, T. Wallerath, C. Euchenhofer, I. Ihrig-Biedert, H. Li, U. Forstermann, Hypertension 31 (1998) 582^588. [17] N. Inoue, R.C. Venema, H.S. Sayegh, Y. Ohara, T.J. Murphy, D.G. Harrison, Arterioscler. Thromb. Vasc. Biol. 15 (1995) 1255^1261. [18] M. Yoshizumi, M. Perrella, J.J. Burnett, M. Lee, Circ. Res. 73 (1993) 205^209. [19] L.P. McQuillan, G.K. Leung, P.A. Marsden, S.K. Kostyk, S. Kourembanas, Am. J. Physiol. 267 (1994) H1921^H1927. [20] M.A. Flowers, Y. Wang, R. Stewart, B. Patel, P.A. Marsden, Am. J. Physiol. 269 (1995) H1988^H1997. [21] J. Liao, W. Shin, W. Lee, S. Clark, J. Biol. Chem. 270 (1995) 319^324.
BBAEXP 91215 10-12-98
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