Hypoxia stimulates human preproendothelin-1 promoter activity in transgenic mice

Catherine R. Aversa1, Suzanne Oparil2, Jaime Caro3, Huaibin Li2, Shuang-Dan Sun2, Yiu-Fai Chen2, Mavis R. Swerdel4, Thomas M. Monticello5, Stephen K. Durham5, Alexander Minchenko3, Sergio A. Lira4, and Maria L. Webb1

Departments of 1 Cardiovascular Biochemistry, 5 Experimental Pathology, and 4 Oncology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543; 2 Vascular Biology and Hypertension Program, University of Alabama at Birmingham, Birmingham, Alabama 35294; and 3 Department of Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Significant elevations in endothelin (ET)-1 levels accompany many diseases, but the underlying regulatory mechanisms are unclear. To investigate the in vivo regulation of human preproendothelin-1 (PPET-1), we examined the activity of the PPET-1 promoter in transgenic mice exposed to hypoxia. Mice expressing one of three PPET-1 promoter-luciferase (PPET-1/LUC) reporter transgenes (approx 2.5 kb, 138 bp, or none of the 5'-flanking sequences of the PPET-1 gene) were generated. LUC expression was reduced in mice with a truncated 138-bp PPET-1 promoter. Exposure of mice bearing the 2.5-kb PPET-1/LUC transgene to hypoxia (10% O2 for 24 h) increased LUC expression sixfold in pulmonary tissue but only twofold in other tissues. In situ hybridization revealed the strongest transgene expression in the pulmonary vasculature and bronchiolar epithelium. These data are consistent with the hypothesis that hypoxic induction of the PPET-1 gene leads to increased pulmonary production of ET-1 in diseases associated with low O2 tension.

endothelin; heart failure; pulmonary hypertension

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE ENDOTHELIN (ET) peptides (ET-1, ET-2, and ET-3) comprise a family of structurally and pharmacologically related agents with potent biological activity, including contraction and vasodilation of smooth muscle, pressor and depressor effects in the renal vasculature, and positive myocardial inotropy and chronotropy (47). These biologically active peptides arise from specific preproendothelin (PPET) proteins, each of which is encoded by a separate gene. ET-1 was originally isolated from porcine endothelial cells (46), but it has since been shown to be produced by vascular smooth muscle cells (37), respiratory epithelial cells (38), renal glomerular, epithelial and mesangial cells (6, 19), keratinocytes (48), and neurites (26, 30). Significant elevations in plasma ET-1 accompany pulmonary hypertension (10), congestive heart failure (CHF) (4, 21, 27), cardiogenic shock (5), systemic hypertension (20), and myocardial ischemia (32, 35, 43), suggesting that ET-1 production and/or hydrolysis are altered in these conditions. ET receptors are also affected by ischemic disease. Nambi et al. (34) observed an altered affinity of ET receptors for 125I-labeled ET-1 after renal ischemia, and Bird et al. (2) reported that ETA receptor density is increased in kidneys from spontaneous hypertensive rats compared with normotensive rats. The actions, associations, and responsiveness of the components of the ET receptor system to physiological and pathophysiological stimuli has led to the suggestion that ET may be causally involved in a variety of disease processes.

Attempts to define a role for ET in disease have employed various inhibitors of ET action, including receptor antagonists, anti-ET antibodies, and inhibitors of endothelin-converting enzyme. Several antagonists have significantly attenuated neointima formation after balloon injury of the rat carotid artery (7, 9). Kowala et al. (23) demonstrated that the ETA subtype-selective antagonist BMS-182874 decreased atherosclerotic lesion size in hamsters fed high-cholesterol diets compared with vehicle-treated control animals. Balanced ET receptor antagonism with bosentan had beneficial effects on hemodynamic and cardiac parameters in patients with class III heart failure (18). In ischemic myocardial disease, anti-ET-1 antibodies (43), phosphoramidon, a nonspecific endothelin-converting enzyme inhibitor, and ETA receptor antagonists (13, 14) protected ischemic hearts in vivo. These data are consistent with an emerging role for ET in a variety of vascular diseases.

Another approach to elucidation of the role of ET in disease is through transgenic animals in which the PPET genes are either overexpressed or ablated through homologous recombination. Recent work with mice rendered deficient in ET-1 through homologous recombination has shown that ET-1 is essential for normal development and cardiovascular homeostasis (24). Puffenberger and colleagues (36) have also shown that a missense mutation in the ETB receptor gene is involved in Hirschsprung's disease, a syndrome characterized by altered innervation of the distal colon. Moreover, targeted or natural disruption of the ETB gene (17) or targeted disruption of the ET-3 gene (12) mimics Hirschsprung's disease. In contrast, no successful attempts to overexpress the PPET-1 gene have been reported.

Despite significant elevations in circulating and tissue levels of ET in diseases of low O2 tension, such as pulmonary hypertension and CHF, the regulatory mechanisms underlying disease-associated increases in ET-1 production remain unclear. Studies have shown that PPET-1 mRNA and protein levels increase in cultured human endothelial cells exposed to hypoxia (22) and in pulmonary vascular endothelial cells of patients with primary pulmonary hypertension (10). Studies in rats have shown that exposure to hypoxia increases PPET-1 mRNA levels in lungs and pulmonary arteries (8, 28). To begin to elucidate the in vivo regulation of human PPET-1 gene expression in pulmonary hypertension and CHF, transgenic mice that express the firefly luciferase (LUC) reporter gene under the control of the human PPET-1 promoter were developed. In the present study, we show that pulmonary-specific enhancement of the human PPET-1 promoter activity occurs in vivo in response to hypoxia.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
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References

Subcloning and purification of the PPET-1/LUC constructs. A 18-kb genomic fragment containing the full-length human PPET-1 gene was generously provided by Dr. Tom Quertermous (Vanderbilt University; see Ref. 3). For construction of a plasmid containing 2.459 kb of 5'-flanking sequences, a 2.6-kb Xho I/Bgl II PPET-1 fragment was subcloned into the Xho I/BamH I site of the LUC expression vector KSbLUC. The resulting plasmid, PPET-1/LUC(-2459), contains several cis-acting regulatory elements upstream from the transcription initiation site (Fig. 1). A second plasmid, containing only 138 bp of 5'-flanking sequence PPET-1/LUC(-138), was created by subcloning a 308-bp Bsg I/Bgl II fragment into the Sma I/Bgl II sites of KSbLUC expression vector. This construct removes most of the 5' regulatory elements but retains the GATA site as well as the TATA and CAAT boxes. For transfection experiments, DNA was purified from agarose gels using the Qiagen Plasmid Purification Kit, according to the manufacturer's instructions (Qiagen, Chatsworth, CA).


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Fig. 1.   Schematic diagram of preproendothelin (PPET)-1/luciferase (LUC) constructs used for transfection experiments and the generation of transgenic mice. A 2624- or 303-bp fragment excised from the human PPET-1 gene was ligated to the LUC expression vector KSbLUC to create PPET-1/LUC(-2459) and PPET-1/LUC(-138), respectively. APR, acute phase response element; AP-1, activator protein 1, fos/jun binding site; NF-1, nuclear factor (NF)-kappa B-1 binding site. GATA, CACAAT, and TATAAA are conserved motifs typically found in or near promoters.

In vitro expression of PPET/LUC DNAs. Before injection into oocytes, plasmid DNA containing fusion gene constructs was transfected into bovine pulmonary artery endothelial (BPAE) cells (American Type Culture Collection, Rockville, MD). Confluent flasks of BPAE cells were seeded into 35-mm plates and were allowed to incubate overnight to 50% confluence. Cells were then transfected with approx 3 µg of PPET-1/LUC(-2459), PPET-1/LUC(-138), and KSbLUC DNA for 18 h using Lipofectin reagent, according to the manufacturer's instructions (Life Technologies, Gaithersburg, MD). Mock transfected cells incubated with Lipofectin reagent without DNA and untreated cells were also tested. After 48 h, cells were rinsed with phosphate-buffered saline (PBS) and were lysed in 1× reporter lysis buffer (Promega, Madison, WI). Lysates were centrifuged at 13,000 revolutions/min (rpm) for 30 s. Supernatants were transferred to new tubes and either assayed immediately or stored at -80°C.

Production and characterization of transgenic mice. The PPET-1 promoter/LUC constructs, including SV40 polyadenylation sequences PPET-1/LUC(-2459), PPET-1/LUC(-138), and LUC, were excised from the KSbLUC vector and were purified by sucrose gradient centrifugation. The purified DNA constructs were microinjected into the male pronucleus of fertilized mouse eggs (C57BL/6J × DBA/2F2; Jackson Laboratory, Bar Harbor, ME). Microinjected eggs were then transferred into the oviducts of Institute for Cancer Research foster mothers (Harlan Sprague Dawley, Indianapolis, IN). Identification of transgene-positive mice was conducted by polymerase chain reaction (PCR) analysis of mouse genomic DNA with two sets of primers: one that amplifies a 359-bp fragment of the firefly LUC gene and a second set that amplifies a 490-bp fragment of the endogenous ZP3 control gene (29).

RNA analysis. Mouse tissues were excised, immediately frozen in liquid N2, and stored at -80°C until processing. Total RNA was isolated using the UltraSpec RNA Isolation System, according to the manufacturer's instructions (Biotecx, Houston, TX). Precipitated RNA was resuspended and stored in formamide. RNA yield was quantitated by spectrophotometry and was confirmed by agarose gel electrophoresis with ethidium bromide staining. Thirty and three micrograms of RNA were applied to a Nytran membrane (Schleicher & Schuell, Keene, NH) by vacuum blotting using a slot-blot manifold (Hoefer Scientific, San Francisco, CA). Hybridization was performed in 50% formamide, 5× sodium chloride-sodium phosphate-EDTA (SSPE), 1× Denhardt's solution, 1% sodium dodecyl sulfate (SDS), and 100 µg/ml sheared herring testes DNA at 42°C. A 559-bp probe that hybridizes to a sequence in the 3'-untranslated region of the PPET-1 mRNA was labeled by random priming using the RediPrime System, according to the manufacturer's instructions (Amersham, Arlington Heights, IL), and was added to the hybridization solution. After overnight incubation, the slot blot was washed two times with 2× SSPE and 0.1% SDS at 42°C and two times with 0.5 SSPE and 0.1% SDS at 60°C. The relative intensity of each slot was quantitated using a PhosphorImager (Molecular Devices, Sunnyvale, CA), and data quantitation was conducted with ImageQuant image analysis software.

LUC assays of cell and tissue extracts. Organs excised from mice were immediately frozen in liquid N2 and were then sonicated in a solution containing 25 mM glycylglycine (pH 7.8), 15 mM MgSO4, 1 mM dithiothreitol (DTT), and 1% Triton X-100. Lysates were centrifuged at 12,000 rpm in a microfuge for 30 min at 4°C. Supernatants were transferred to another tube and were stored at -80°C until assay. Extracts from cells and tissues were assayed using the Luciferase Assay System, according to the manufacturer's instructions (Promega). Ten (cells) or twenty-five (tissues) microliters of lysate were injected with one hundred microliters of luciferase assay reagent [20 mM Tricine, 1.07 mM (MgCO3)4Mg(OH)2-5H2O, 0.1 mM EDTA, 33.3 mM DTT, 270 µM CoA, 470 µM luciferin, and 530 µM ATP]. Light emissions were quantitated for 30 s at 25°C in a luminometer (EG & G Berthold Automat LB 953, Princeton, NJ).

Hypoxia treatment. To investigate the activity of the PPET-1 promoter under low O2 conditions, the effect of hypoxia on transgene expression was examined. Mice at 8-10 wk of age were exposed for 24 h to room air or normobaric hypoxic (10 ± 0.5% O2) conditions in a box in which CO2 was kept at <0.2% and relative humidity was kept at <70% as previously described (28). Immediately after the 24-h exposure period, tissues were harvested for LUC assay.

In situ hybridization. At the end of the 24-h hypoxic exposure, mice were removed individually from the hypoxic chamber or from their cages and were rapidly killed by decapitation. The lungs were excised quickly, rinsed in cold PBS solution, and fixed in 4% paraformaldehyde for 3 h and in 15% sucrose overnight at 4°C. Frozen samples were embedded in HistoPrep frozen tissue-embedding media (Fisher Scientific, Pittsburgh, PA) on dry ice and were stored at -80°C.

The LUC probe was generated by amplifying a 249-bp region of the LUC gene contained in the KSbLUC vector with PCR primers that incorporated a Sac I site on the 5'-end and a Hind III site on the 3'-end of the fragment. The fragment was then restriction digested and subcloned into corresponding sites in pGEM4z. The LUC-pGEM4z plasmid was linearized using restriction enzymes Sac I or Hind III and was used as a template for transcription of antisense and sense cRNA probes. Antisense and sense cRNA probes specific for LUC mRNA and for murine PPET-1 mRNA were synthesized in vitro using a Riboprobe Gemini Core Kit with T7 or SP6 RNA polymerase, according to the manufacturer's instructions (Promega), in the presence of [33P]UTP (NEN, Wilmington, DE). The specific activity of each probe was ~1-2 × 108 counts · min-1 (cpm) · µg DNA-1. The murine PPET-1 gene was generously provided by Dr. Koji Maemura (University of Tokyo).

In situ hybridization was conducted using a standard protocol on frozen sections (44). Ten-micrometer cryostat sections were prepared and transferred to VectorBond-coated slides (Vector Labs, Burlingame, CA). Sections were postfixed for 10 min in freshly prepared 4% paraformaldehyde at 4°C, washed in 0.5× standard sodium citrate (SSC), digested with proteinase K (1-5 µg/ml, 10 min, room temperature), washed with 0.5× SSC, and prehybridized with 100 µl of hybridization buffer [20 mM tris(hydroxymethyl)aminomethane (pH 7.5), 5 mM EDTA, 0.3 M NaCl, 10 mM DTT, 1× Denhardt's solution, 10% dextran sulfate, and 50% formamide] in an atmosphere equilibrated with 4× SSC and 50% formamide at 42°C for 1-3 h. Approximately 6-10 × 108 cpm of the 33P-labeled antisense or sense cRNA probes described above were used for each tissue section. The riboprobe and 50 µg of tRNA were denatured at 100°C for 3 min, diluted rapidly in ice-cold hybridization buffer, added directly to the tissue overlay, and incubated at 55°C overnight. The slides were washed twice with 2× SSC containing 10 mM beta -mercaptoethanol (beta -ME) and 250 mM EDTA for 10 min at room temperature, treated with 20 µg/ml ribonuclease A for 30 min, and then washed twice in 2× SSC with beta -ME-EDTA. High-stringency washes, initially at 55°C for 2 h in 0.1× SSC, 10 mM beta -ME, and 1 mM EDTA, were followed by two brief washes in 0.5× SSC. Sections were then dehydrated through a graded series of ethanol (50%, 70%, and 90%) containing 0.3 M ammonium acetate. Slides were dried in a vacuum dessicator, dipped in photographic emulsion (Kodak NTB2) diluted 1:1 with distilled water, dried in the dark for 2 h, and exposed on a light-tight box at 4°C with dessicant for 2-6 wk. The emulsion-coated slides were developed and counterstained with hematoxylin and eosin and were examined by bright-field microscopy.

Statistical analysis. Statistical significance was determined using analysis of variance or two-tailed Student's t-test. Significant F-values were evaluated with a pairwise test of individual group means using Bonferroni probabilities.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

In vitro expression of PPET-1/LUC DNAs. To determine if the PPET-1/LUC(-2459) and PPET-1/LUC(-138) DNAs were expressed in vitro, BPAE cells were transiently transfected with plasmid DNA containing these constructs. Comparison of LUC activities in the PPET-1/LUC(-2459) and PPET-1/LUC(-138) transfected cells demonstrates that, while both constructs confer LUC expression on otherwise null cells, the truncated promoter drives LUC expression at an approximately eightfold lower levels than does the 2.5-kb promoter and 5'-flanking sequences. Cells transfected with the KSbLUC vector had negligible LUC activity (Fig. 2). Equivalent transfection efficiencies were confirmed by cotransfection in the presence of a beta -galactosidase reporter vector (data not shown). This finding is consistent with the suggestion that regulatory elements in the 5'-sequence upstream of -138 bp facilitate PPET-1 expression.


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Fig. 2.   Expression of LUC reporter gene constructs in bovine pulmonary artery endothelial cells transfected with PPET-1/LUC(-2459), PPET-1/LUC(-138), or KSbLUC. Transfections were conducted using Lipofectin reagent as described in MATERIALS AND METHODS. Mock transfected cells (MOCK) were incubated with transfection reagent in the absence of DNA. An eightfold increase in LUC production was observed in cells transfected with PPET-1/LUC(-2459) when compared with PPET-1/LUC(-138). * Significant difference from PPET-1/LUC(-2459) at P < 0.05.

In vivo expression of PPET-1/LUC DNAs. After excision of the transgenes from the KSbLUC vector, transgenic founders were generated expressing the PPET-1/LUC(-2459), PPET-1/LUC(-138), and LUC constructs (Fig. 1). Of the animals tested, 10 positives were identified for PPET-1/LUC(-2459). In addition, 10 positives were identified for PPET-1/LUC(-138). The PPET-1/LUC(-138) mice were used for validation of promoter expression. In addition, eight positives were identified for LUC (data not shown).

Validation of PPET-1 promoter activity. To assess if the PPET-1/LUC(-2459) transgene was expressed in a physiologically relevant manner, the tissue pattern of LUC activity in PPET-1/LUC(-2459) transgenic mice was compared with endogenous PPET-1 mRNA expression in normal mice. PPET-1 mRNA was detected in all tissues examined, and the presence of PPET-1 mRNA correlated with the presence of LUC enzyme activity (Table 1). These data demonstrate that the PPET-1/LUC(-2459) transgene is expressed in the same tissues as endogenous ET-1.

                              
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Table 1.   Comparison of endogenous PPET-1 mRNA expression to LUC activity in tissues from PPET-1/LUC(-2459) mice

As shown in Fig. 2, truncation of the PPET-1 promoter reduced promoter activity in vitro. To determine whether truncation of the PPET-1 5'-flanking sequence affected gene expression in vivo as it had in vitro, the level of PPET-1 promoter-directed LUC expression was analyzed in each of three transgenic lines varying only in the extent of 5'-flanking sequence. In all tissues examined, LUC activity in both the PPET-1/LUC(-2459) and PPET-1/LUC(-138) mice was elevated over that in the promoterless LUC control animals, in which LUC activity was negligible (Fig. 3). Moreover, a marked 10- to 30-fold increase in LUC activity was observed in most tissues derived from the PPET-1/LUC(-2459) mice compared with those derived from PPET-1/LUC(-138) mice (Fig. 3). These data indicate that the PPET-1 promoter drives LUC expression in the same tissues as PPET-1 mRNA is found and with similar promoter strength as seen in vitro. In addition, the data suggest that tissues such as lung, heart, kidney, liver, and spleen require additional regulatory elements upstream of the -138-bp region of PPET-1 for full expression. In contrast, PPET-1/LUC expression in aorta was similar in PPET-1/LUC(-2459) and PPET-1/LUC(-138) mice.


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Fig. 3.   Analysis of LUC activity in tissues from transgenic progeny. PPET-1/LUC(-2459), PPET-1/LUC(-138), or LUC transgenes were excised from the KSbLUC vector and were used for the generation of transgenic mice. LUC activity is reported as average relative light units (RLU) + SE from n = 5-6 mice. * Differences at P < 0.05 compared with PPET-1/LUC(-2459).

Effect of hypoxia on PPET-1 promoter activity. LUC activity was assessed in PPET-1/LUC(-2459) mice exposed for 24 h to room air or hypoxic (10% O2) conditions. Hypoxia significantly stimulated LUC enzyme activity in the lung, heart, kidney, liver, and spleen (Fig. 4A). The greatest induction was observed in pulmonary tissue, in which hypoxia stimulated a sixfold increase in LUC over room air controls. Hypoxic induction of LUC was twofold over control in heart, kidney, liver, and spleen. There was no stimulation of LUC activity in the tail (data not shown). Verification that hypoxic induction of LUC activity is mediated via the PPET-1 promoter was demonstrated by the absence of LUC activity in promoterless LUC mice after exposure to 10% O2 for 24 h (Fig. 4B). These data demonstrate that PPET-1 promoter activity is stimulated by hypoxia in a pulmonary-specific manner.


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Fig. 4.   Effect of hypoxia on LUC activity in mice expressing the human PPET-1/LUC(-2459) construct (A) or a promoterless LUC construct (B). Mice expressing the PPET-1/LUC(-2459) transgene were exposed to room air or hypoxic (10 ± 0.5% O2 for 24 h) conditions before death (see MATERIALS AND METHODS). PPET-1 promoter activity was assessed by LUC reporter gene expression. LUC activity is reported as degree of increase + SE from hypoxic tissues compared with normoxic tissue from n = 6 mice. * Significant difference at P < 0.05 compared with air control.

Cellular localization of PPET-1/LUC transgene expression. The cellular localization of PPET-1/LUC(-2459) expression in lung from hypoxia-exposed transgenic mice was determined using in situ hybridization with an antisense LUC probe. In lungs of hypoxic mice, strong hybridization was detected in pulmonary vessels in both smooth muscle and endothelial cells (Fig. 5A). Expression of the transgene was not confined to vascular tissue, as antisense hybridization was also detected in pulmonary bronchiolar epithelial cells and alveoli (Fig. 5A). Diffuse hybridization was also observed in the lung parenchyma. The hybridization patterns were similar, but substantially lower, in normoxic mice (data not shown). Hybridization of the sense LUC probe to hypoxic and normoxic mice yielded diffuse and nonspecific staining (Fig. 5C).


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Fig. 5.   Comparison of LUC mRNA and endogenous PPET-1 mRNA by in situ hybridization in hypoxic pulmonary tissue. Mice were exposed to hypoxic conditions for 24 h as described in MATERIALS AND METHODS. A and C: photomicrographs showing hybridization in hypoxic pulmonary tissue of PPET-1/LUC(-2459) mice to LUC antisense (A) and sense (negative control; C) probes. Grains observed with the antisense probe are localized to endothelial and smooth muscle cells of the pulmonary vessel and to epithelial cells of the pulmonary bronchiole. B and D: photomicrographs showing hybridization in normal mouse pulmonary tissue to a mouse PPET-1 antisense probe (B) and sense (negative control; D) probes. Grains observed with the antisense probe are localized to bronchiolar epithelial cells, vascular endothelial cells, alveolar septa, and, to a lesser extent, smooth muscle cells. The localization of PPET-1 antisense probe is similar to that of PPET-1/LUC(-2459). b, Bronchiole; v, vessel. Hematoxylin and eosin stain; magnification, ×400.

To demonstrate that cellular expression of LUC is under the control of the PPET-1 promoter, we correlated the cellular localization of PPET-1/LUC(-2459) to the endogenous cellular expression of murine PPET-1. In situ hybridization was conducted in lungs of hypoxic mice using a murine PPET-1 cRNA probe. Intense hybridization was detected in hypoxic mouse lungs with the antisense but not the sense probe (Fig. 5, B and D). Hybridization with the antisense murine PPET-1 probe yielded a strong signal in arteriole endothelium and a weak signal in the smooth muscle layer. As with the LUC hybridizations, a hybridization signal was also obtained in pulmonary bronchiolar epithelial cells and alveoli (Fig. 5B). Hybridization with the sense PPET-1 probe yielded diffuse and nonspecific staining (Fig. 5D).

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

To elucidate the in vivo regulation of human PPET-1 gene expression in pulmonary hypertension and CHF, transgenic mice that express the firefly LUC reporter gene under the control of the human PPET-1 promoter were developed. The dynamic range of the LUC reporter against a null background in mice affords a much more sensitive in vivo assay to assess promoter activity than measuring PPET-1 mRNA. In the present study, we show that pulmonary-specific enhancement of the human PPET-1 promoter activity occurs in vivo in response to hypoxia.

Several lines of evidence demonstrate that the PPET-1/LUC(-2459) transgene is expressed in a physiologically relevant manner. First, the tissue pattern of PPET-1 transgene expression is consistent with the endogenous mouse PPET-1 mRNA expression pattern observed here and reported elsewhere. Sakurai et al. (40) studied endogenous PPET-1 expression in the rat and found strong hybridization in heart, lung, kidney, spleen, submandibular gland, liver, and stomach. Hilkert et al. (16) studied endogenous PPET-1 mRNA expression in mice and determined that lung, brain, kidney, spleen, and heart were positive tissues. Additional studies have since demonstrated the presence of ET-1 transcripts in bladder (39) and human skin (48). Thus the data reported here for expression of the PPET-1/LUC transgene are consistent with previous observations of the endogenous expression of PPET-1 mRNA.

A second line of evidence in support of the physiological regulation of the PPET-1/LUC(-2459) transgene derives from studies with a truncated promoter. Wilson and colleagues (45) previously observed that deletion of the 5'-flanking sequence of human PPET-1 affected expression of a growth hormone (GH) reporter gene. Constructs containing the proximal 157 bp of the PPET-1 promoter exhibited near maximal expression of GH, whereas truncations beyond -157 bp significantly attenuated GH production. We observed an eightfold decrease in promoter activity in cells transfected with a promoter truncated at -138 bp. Similarly, in vivo expression of the PPET-1/LUC transgene with only 138 bp of 5'-flanking sequence is 10- to 30-fold reduced in most tissues. The observation that expression of PPET-1/LUC(-2459) and PPET-1/LUC(-138) was the same in aorta suggests that additional cis-acting regulatory elements not present in the 2.5-kb upstream region of the PPET-1 gene are required for full expression in this tissue. Regulatory elements present in the 5'-region include a consensus sequence for an acute phase regulatory element thought to mediate the induction of mRNA under acute physical stress, two activator protein (AP)-1/JUN- or 12-O-tetradecanoylphorbol-13-acetate-responsive elements known to modulate transcriptional activation in many mammalian systems, and a nuclear factor-1 consensus sequence that mediates transcriptional activation by transforming growth factor-beta . Alternatively, these tissues may not express the complement of transcription factors that interact with the region of the 5'-flanking sequence spanning -138 to -2459 bp.

In situ hybridization with specific antisense LUC and mouse PPET-1 probes in hypoxic PPET/LUC(-2459) mice demonstrates colocalization of these mRNAs. We observed expression of LUC and PPET-1 mRNA in pulmonary vessels, bronchiolar epithelium, and alveoli. Interestingly, although high levels of LUC expression were detectable in both the endothelium and smooth muscle of pulmonary vessels, PPET-1 expression was mostly in the endothelium. This difference in cellular localization could be due to the absence of additional cis-acting elements in the PPET/LUC(-2459) construct that may confer cell specificity. Our data are consistent with previous studies that examined the patterns of PPET-LUC transgene expression in mice. Immunostaining with anti-LUC antibodies has been previously observed in murine bronchiolar epithelium (15). These investigators observed that a 2.4-kb segment of 5'-flanking sequence, the first exon of the gene and 0.8 kb of the first intron, directed LUC transgene expression to the endothelium of large and small vessels as well as to arterial smooth muscle and select epithelial surfaces. Using a 4.3-kb segment of the human PPET-1 5'-flanking and promoter sequence, Hilkert et al. (16) demonstrated a cell-specific expression pattern of the transgene in endothelial cells of blood vessels and epithelial cells of the lung.

Significant elevations in circulating and tissue ET levels accompany numerous cardiovascular diseases. The present data suggest that the 2.5-kb 5'-flanking sequence of the human PPET-1 gene contains a hypoxia-responsive element that is activated in a pulmonary-specific manner. These findings are consistent with the suggestion that in diseases associated with low O2 tension, such as pulmonary hypertension and CHF, hypoxic activation of the PPET-1 gene leads to increased pulmonary production of ET-1. The responsiveness of the human PPET-1/LUC(-2459) transgene to hypoxia is consistent with previous accounts of increased plasma levels of ET-1 and steady-state PPET-1 mRNA levels after exposure to hypoxia. Acute (24-48 h) or chronic (4 wk) exposure of rats to hypoxic conditions elevated plasma ET-1 and also caused a selective increase in pulmonary PPET-1 mRNA levels as little or no increase in PPET-1 mRNA was observed in organs perfused by systemic circulation (8, 28, 42). Thus the present observation of greater induction of LUC activity in lung compared with other tissues confirms and extends these earlier observations. In humans, plasma ET-1 levels increase progressively with the hypoxia associated with high altitude (33). This increase in plasma levels is likely the result of increased PPET-1 gene transcription as Kourembanas and colleagues (22) showed that hypoxia increases PPET-1 gene transcription rates in cultured human endothelial cells. Taken together, these data suggest that the elevations in circulating ET-1 observed in hypoxia are due primarily to increased pulmonary PPET-1 gene transcription.

The mechanism for the hypoxic induction of the PPET-1 gene is unclear. Previous studies with hypoxia-sensitive genes such as erythropoietin (EPO) and vascular endothelial growth factor (VEGF) (1, 11, 25, 31) suggest that a hypoxia-responsive enhancer (HRE) and hypoxia-inducible factor-1 (HIF-1) are involved. Analysis of the 5'-flanking sequence of the PPET-1 gene shows that a 7-bp-long motif similar to the core HIF-1 binding site of the EPO and VEGF genes (41) exists at position -117 to -124. We speculate that the PPET-1 gene belongs to the set of hypoxia-inducible genes and that the lung contains greater quantities of HIF-1 available for interaction with the HRE in PPET-1 than other organs such as heart or kidney. Additional studies are necessary to test this hypothesis and to determine if hyperoxia has a similar or different effect on PPET-1 transcription.

In summary, the data presented here provide evidence for pulmonary-specific hypoxic induction of the PPET-1 promoter and support the hypothesis that pulmonary production of ET-1 contributes to diseases associated with low O2 tension such as pulmonary hypertension and CHF. The PPET-1/LUC transgenic mice described here will be useful in delineating this mechanism and role of ET-1 production during the course of specific disease progression.

    ACKNOWLEDGEMENTS

We extend thanks to Dr. Koji Maemura of the University of Tokyo for the murine PPET-1 gene. We also gratefully acknowledge the expertise of Petronio Zalamea for oocyte injection and thank the Veterinary Science Department of Bristol-Myers Squibb and the University of Alabama for scientific expertise and animal care during the course of these studies.

    FOOTNOTES

Address for reprint requests: M. L. Webb, Pharmacopeia Inc., 3000 Eastpark Blvd, Cranbury, NJ 08512.

Received 4 February 1997; accepted in final form 10 July 1997.

    REFERENCES
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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