ACCELERATED COMMUNICATION Effect of an Antisense Oligodeoxynucleotide to Endothelin- Converting Enzyme-1c (ECE-1c) on ECE-1c mRNA, ECE-1 Protein and Endothelin-1 Synthesis in Bovine Pulmonary Artery Smooth Muscle Cells

Endothelin-1 (ET-1) is secreted from endothelial and vascular smooth muscle cells (VSMC) after intracellular hydrolysis of big ET-1 by endothelin converting enzyme (ECE). The metallopeptidase called ECE-1 is widely thought to be the physiological ECE, but unequivocal evidence of this role has yet to be provided. Endothelial cells express four isoforms of ECE-1 (ECE1a, ECE-1b, ECE-1c, and ECE-1d), but the identity of ECE-1 isoforms expressed in VSMC is less clear. Here, we describe the characterization of ECE-1 isoforms in bovine pulmonary artery smooth muscle cells (BPASMC) and the effect on ET-1 synthesis of an antisense oligodeoxynucleotide (ODN) to ECE1c. Reverse transcriptase-polymerase chain reaction (RT-PCR) evaluation of total RNA from BPASMC showed that ECE-1a and ECE-1d were not expressed. Sequencing of cloned ECE-1 cDNA products and semiquantitative RT-PCR demonstrated that ECE-1b and ECE-1c were expressed in BPASMC, with ECE-1c being the predominant isoform. Basal release of ET-1 from BPASMC was low. Treatment for 24 h with tumor necrosis factor-a (TNFa) stimulated ET-1 production by up to 10-fold with parallel increases in levels of preproET-1 mRNA. Levels of ECE-1c mRNA were also raised after TNFa, whereas amounts of ECE-1b mRNA were decreased significantly. Treatment of BPASMC with a phosphorothioate antisense ODN to ECE-1c caused a marked reduction in ECE-1c mRNA levels and ECE-1 protein levels. However, basal and TNFa-stimulated ET-1 release were largely unaffected by the ECE-1c antisense ODN despite the inhibition of ECE-1c synthesis. Hence, an endopeptidase distinct from ECE-1 is mainly responsible big ET-1 processing in BPASMC. Endothelin-1 (ET-1) is derived from its precursor, preproendothelin-1, by intracellular proteolytic processing (Corder et al., 1995; Harrison et al., 1995; Woods et al., 1999). The final step in its biosynthesis involves specific enzymatic hydrolysis of the intermediate, big ET-1, by an endothelinconverting enzyme (ECE) (Yanagisawa et al., 1988). ET-1 exerts wide-ranging effects on a variety of tissues and cell types through interaction with two subtypes of cell surface receptors (ETA and ETB receptors) (Douglas, 1997; Haynes and Webb, 1998). It has been implicated as a causative factor in the pathogenesis of hypertension, pulmonary hypertension, congestive heart failure, atherosclerosis, and asthma (Douglas, 1997; Haynes and Webb, 1998; Goldie and Henry, 1999). A number of highly potent ET receptor antagonists have been developed for therapeutic use. These compounds are generally selective for ETA receptors or nonselective ETA/ ETB antagonists (Douglas, 1997). In some tissues, most notably the airways, ETB receptors predominate yet they are resistant to blockade by selective ETB or nonselective ETA/ ETB receptor antagonists (Hay et al., 1998). Therefore, specific inhibition of ET-1 synthesis with ECE inhibitors may be a better approach for attenuating the adverse effects of ET-1 excess under some conditions. This work was supported by the Institut de Recherches Internationales Servier and The William Harvey Research Foundation. ABBREVIATIONS: ET-1, endothelin-1; ECE, endothelin converting enzyme; ODN, oligodeoxynucleotide; BPASMC, bovine pulmonary artery smooth muscle cells; DMEM, Dulbecco’s modified Eagle medium; BAEC, bovine aortic endothelial cells; RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction; RT, reverse transcriptase; bp, base pair(s); TNFa, tumor necrosis factor a; VSMC, vascular smooth muscle cells. 0026-895X/01/5902-163–169$3.00 MOLECULAR PHARMACOLOGY Vol. 59, No. 2 Copyright © 2001 The American Society for Pharmacology and Experimental Therapeutics 511/880960 Mol Pharmacol 59:163–169, 2001 Printed in U.S.A. 163 at A PE T Jornals on A uust 7, 2017 m oharm .aspeurnals.org D ow nladed from Two endothelin-converting enzyme genes have been cloned and are referred to as ECE-1 (Schmidt et al., 1994, Shimada et al., 1994; Xu et al., 1994) and ECE-2 (Emoto and Yanagisawa, 1995). ECE-1 is the most extensively studied of these two endopeptidases. It is widely expressed in many cells and tissues (Korth et al., 1999). ECE-1 was originally thought to be expressed as two isoforms, ECE-1a and ECE-1b (Valdenaire et al. 1995). More recent findings, however, have revealed two additional isoforms: ECE-1c (Schweizer et al., 1997) and ECE-1d (Valdenaire et al., 1999). The four ECE-1 isoforms result from alternative splicing at the 59-end of a single gene (Schweizer et al., 1997; Valdenaire et al., 1999). They share a common C-terminal region (encoded by exons 4–19), that includes a transmembrane domain and the enzyme catalytic site. Studies of the distribution of the four isoforms have shown ECE-1c to have the highest relative expression (Schweizer et al., 1997; Valdenaire et al., 1999). Based on gene deletion studies, both ECE-1 and ECE-2 have been proposed as physiologically relevant enzymes for ET-1 biosynthesis (Yanagisawa et al., 1998; Yanagisawa et al., 2000). This conclusion is derived from observations that ECE-1 gene knockout causes abnormalities in cardiac development very similar to targeted disruption of the genes for ET-1 (Kurihara et al., 1994), or the ETA receptor (Clouthier et al., 1998). Biochemical assessment of the effect of ECE-1 knockout on ET-1 synthesis is difficult to investigate in ECE1 embryos because it results in midgestational lethality. ET-1 levels in gestational day 12.5 embryos show reductions of about 40% in ECE-1 null mice (Yanagisawa et al., 1998, 2000), but this may simply reflect the fact these embryos are not viable. If the physiologically essential ECE is eliminated in the ECE-1 mice, raised tissue levels of big ET-1 would be expected because of inhibition of its processing, but no increases were observed (Yanagisawa et al., 1998). Thus, despite the many reports showing that ECE-1 isoforms are able to convert big ET-1 to the mature ET-1 peptide, it is still unclear whether ECE-1 plays a physiological role in big ET-1 processing. The aim of this study was to examine the role of ECE-1c in ET-1 production by using an antisense oligodeoxynucleotide (ODN) to selectively inhibit ECE-1c synthesis in cultured cells. These studies used bovine pulmonary artery smooth muscle cells (BPASMC) because initial investigations revealed ECE-1c to be the major isoform in these cells, with no expression of ECE-1a or ECE-1d. The results show that ECE-1 has only a minor role in ET-1 synthesis. Experimental Procedures Cell Culture. BPASMC were cultured from fresh bovine pulmonary artery in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) fetal bovine serum by using the explant technique (Corder, 1996). Cultured cells, used between passages 5 and 10, exhibited characteristic smooth muscle cell morphology and stained positively for a-actin. Bovine aortic endothelial cells (BAEC) were cultured as described by Corder and Barker (1999). Initial experiments with BPASMC evaluated the ET-1 response to TNFa (R&D Systems, Minneapolis, MN), and the effects of phosphoramidon (Peptide Institute, Osaka, Japan) on ET-1 and big ET-1 release. For both series of experiments, confluent cultures were incubated with the agents being investigated in serum-free DMEM for 24 h. The conditioned media were collected for immunoassay of ET-1 and big ET-1. Characterization of ECE-1 Isoforms Expressed in BAEC and BPASMC by RACE and Colony Hybridization. Total RNA was extracted from confluent BAEC or BPASMC monolayers by using RNAzol B (Biogenesis, Poole, UK). Poly(A) mRNA for BAEC and BPASMC was obtained from total RNA using poly(A) tract oligo dT-magnetic beads (Promega, Southampton, UK). After firstand second-strand synthesis and adaptor ligation, 59 rapid amplification of cDNA ends (59-RACE) was carried out using the Marathon cDNA amplification protocol (Clontech, Basingstoke, UK). Polymerase chain reaction (PCR) was performed by using the adaptor forward primer 59-CCATCCTAATACGACTCACTATAGGGC-39 (AP1) and a reverse primer, 59-GGCGTTCTTGTCTGGTATTGGA-39, corresponding to a sequence common to all bovine ECE-1 isoforms (Fig. 1). For BAEC and BPASMC, cDNA from these reactions were purified, subcloned into the plasmid TA cloning vector pGEM-T Easy (Promega), used to transform competent JM109 Escherichia coli (Promega), and cultured at 37°C for 14 h. Colony hybridization to identify ECE-1 clones was performed with a 214-bp cDNA probe obtained after purification of the BglI (Promega) digest of the 253-bp ECE-1c PCR product (described below). For hybridization, 25 ng of the cDNA probe was labeled using random hexanucleotides and Klenow fragment (Promega) in a 50-ml reaction volume containing 50 mCi of [a-P]dCTP (Amersham Pharmacia Biotech, Little Chalfont, UK) for 4 h at 37°C. Positive colonies were subcultured and further evaluated by performing PCR and restriction digests. Plasmid DNA samples containing inserts of interest were purified and sequenced by using ABI Prism BigDye Terminator Cycle sequencing in conjunction with an ABI Prism 377 sequencer (PE Biosystems, Warrington, UK). Reverse Transcription Polymerase Chain Reaction. RTPCR measurements of mRNA levels were carried out with reagents from Promega with 100 ng of total RNA per reaction under semiquantitative conditions so that the yield of PCR product was proportional to the quantity of RNA template (Barker et al., 1998; Corder and Barker, 1999). For preproET-1, ECE-1a, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), conditions were as described Fig. 1. A, comparison of the nucleotide sequences for bovine ECE-1b, ECE-1c, and ECE-1d obtained in these studies with published sequences of the corresponding human isoforms. Differences are shown as lower case letters in the bovine sequences. The translation start codon for each isoform is shown in bold print. The positions of RT-PCR primer sequences are underlined. The antisense ODN sequence is shown in a 39 to 59 orientation complementary to the cDNA sequence of ECE-1c. B, comparison of the relative expression by semiquantitative RT-PCR of the four ECE-1 isoforms in BAEC and BPASMC using 200 ng and 400 ng of total RNA, respectively. Markers are FX174 RF DNA/HaeIII fragments from Life Technologies (Paisley, Scotland). 164 Barker et al. at A PE T Jornals on A uust 7, 2017 m oharm .aspeurnals.org D ow nladed from (Corder and Barker, 1999). RT-PCR measurements of mRNA levels for ECE-1b, ECE-1c, and ECE-1d were all performed under the same conditions so that a comparison of their relative expression could be made. Reverse transcription with avian myeloblastosis virus RT (5 U per reaction, 48°C for 45 min), primed with isoform specific primers and oligo dT15 primer (1 mM) was followed by PCR using Tfl polymerase (5 U per reaction) with the following cycling parameters: denaturation for 2 min at 94°C; 25 cycles of 94°C for 30 s, 60°C for 3 0s, and 72°C for 1 min; and a final extension at 72°C for 10 min. Isoformspecific ECE-1 primers were based on published sequences for ECE1c, and sequences obtained during these studies from the cloned 59-ends of ECE-1b and ECE-1d. The primers were: ECE-1b (59CGCTGTCGGCGCTGGGGATG-39, forward),ECE-1c(59-GGAGCGCGCGAGCGATGATG-39, forward), ECE-1d (59-CTTAAGGAGTCCGTGCTGCA-39, forward), with a common reverse primer ECE-1b/ 1c/1d (59-GGCGTTCTTGTCTGGTATTGGA-39). These gave PCR products of 253, 253, and 269 base pairs, which were separated by electrophoresis on ethidium bromide-stained 1–1.2% (w/v) agarose gels. In each case, a single band of the expected size was obtained and quantified using a Gel Doc 1000 system (Bio-Rad, Hercules, CA). Antisense ODN Experiments. The sequence chosen as the antisense ODN target was a 25-bp region around the translation start codon of bovine ECE-1c (Schmidt et al., 1994) (Fig. 1). An ODN primer corresponding to this sequence gives robust amplification of a single product by RT-PCR (Corder and Barker, 1999). By analogy with the human ECE-1b and ECE-1c genes, 16 bp at the 39 end of this sequence would be expected to be common to both bovine ECE-1b and ECE-1c. However, the 39 end of the antisense ODN, which is likely to confer its specificity, is complementary only to ECE-1c. An antisense ODN corresponding to this sequence (59-CTTGTAGGTAGACATCATCGCTCGC-39; antisense ODN) was used with its sense counterpart as a control (59-GCGAGCGATGATGTCTACCTACAAG-39; sense ODN). In some experiments, following recommendations of Stein and Krieg (1997), an additional scrambled control ODN was used with the same base composition as the antisense ODN (59-CTACAGATGCGCTCGCTAGATGTTC-39; scrambled ODN). The antisense, sense, and scrambled ODNs were synthesized as full phosphorothioate ODNs and supplied as the high-performance liquid chromatography-purified products (Eurogentec, Seraing, Belgium). BPASMC were seeded at a density of '5 3 10 cells/well in 6 3 35 mm well plates to reach 70 to 80% confluence at the beginning of the transfection procedure. Treatment of BPASMC with phosphorothioate ODNs (400 nM) was performed with the cationic lipid transfectant Tfx-50 (Promega) at a charge ratio of 3:1 (Tfx-50/ODN) in serum-free DMEM. Pilot experiments showed this was the maximum concentration that could be used without cytotoxicity. After 1 h, medium was replaced with DMEM containing 10% fetal bovine serum. The treatment with ODN was repeated 24 later. After a further 24 h, cells were transferred to serum-free DMEM with or without TNFa (30 ng/ml) to study ET-1 synthesis over the following 24 h. At the end of this period, conditioned media were collected for ET-1 and big ET-1 immunoassay. For RT-PCR, cells were harvested in 1 ml of RNAzol B and stored at 280°C. For immunoblotting and measurement of ECE activity, cells were scraped into ice-cold PBS and centrifuged to generate cell pellets that were stored at 280°C for