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Regulation of Adrenomedullin Gene Transcription and Degradation by the c-myc Gene

Department of Clinical and Molecular Endocrinology, Tokyo Medical and Dental University Graduate School, Tokyo 113-8519, Japan

Address all correspondence and requests for reprints to: Masayoshi Shichiri, M.D., Ph.D., Tokyo Medical and Dental University Medical Hospital, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan. E-mail: mshichiri.cme{at}tmd.ac.jp.

	Abstract

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Adrenomedullin, a vasodilatory peptide originally isolated from pheochromocytoma, is known to regulate cell growth, apoptosis, and migration. Overexpression of the c-myc oncogene has been shown to suppress the mouse adrenomedullin gene via the initiator element. We investigated whether c-myc regulates rat and human adrenomedullin genes because there appears to be no initiator elements in their promoter regions. Transactivation of the human adrenomedullin gene by c-myc was demonstrated using a luciferase reporter construct containing an upstream sequence. Using a panel of isogenic rat fibroblast cell lines with differential c-myc expression obtained by targeted homologous recombination, we found markedly elevated adrenomedullin transcript levels in cells stably overexpressing c-myc but a minimal decrease in two independent cell lines containing a homozygous null deletion of c-myc. Degradation of adrenomedullin mRNA was enhanced by a c-myc transgene, resulting in a relatively reserved increase in cellular secretion of adrenomedullin-like immunoreactivity. These results indicate that c-myc transactivates rat and human adrenomedullin genes and accelerates the degradation rate of adrenomedullin mRNA. However, c-myc is not essential for basal expression of the adrenomedullin gene.

	Introduction

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ADRENOMEDULLIN IS A MULTIFUNCTIONAL regulatory peptide originally isolated from human pheochromocytoma as a potent vasorelaxant/hypotensive peptide (1, 2). Adrenomedullin is widely expressed in a variety of tissues, including fibroblasts (3) and endothelial cells (4). A range of cytokines, growth factors, and hormones are reported to increase adrenomedullin gene expression, including TNF- and -ß, IL-1 and -1ß, dexamethasone, cortisol, aldosterone, retinoic acid, and thyroid hormone (2, 5). However, the molecular mechanisms involved in adrenomedullin gene expression largely remain to be elucidated.

An immediate early response gene, c-myc, encodes a nuclear protein containing a basic helix-loop-helix leucine zipper DNA-binding domain. c-Myc protein, by dimerization with Max, recognizes an E-box recognition site (CACGTG) and several related noncanonical sequences (6, 7, 8, 9). c-Myc is known to induce more than a score of genes, including plasminogen activator inhibitor-1 (10), -prothymosin (11), ECA 39 (12), p53 (13), ornithine decarboxylase (14), cad (15), dihydrofolate reductase (16), and cdc25A (17). However, a recent study has revealed that the expressions of most of the proposed c-myc target genes are unchanged in c-myc-null cell lines (18). Although c-Myc regulates proliferation, mitogenesis, differentiation, and programed cell death (19), it is not essential for cell proliferation (20). However, c-Myc can also repress transcription through the initiator element (21) and, in this manner, may participate in the biphasic regulation of genes, such as the adenovirus-2 major late promoter (22) and, possibly, the preproendothelin-1 gene (23). Recently, c-Myc was reported to suppress mouse adrenomedullin expression via an initiator element (24). However, there is no known initiator element in rat or human adrenomedullin promoter sites, and thus, the influence of c-Myc on adrenomedullin expression in these species remains unknown.

In this study, we used a panel of isogenic fibroblast cell lines with differential c-myc expression levels obtained by targeted homologous recombination. c-myc-null cells have prolonged G₁ and G₂ phases with a markedly extended cell cycle, but they still continue to grow (20). The present study was designed to elucidate the regulation of rat and human adrenomedullin expression by physiological and deregulated c-Myc levels.

	Materials and Methods

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Plasmids
A human adrenomedullin promoter construct, designated phADM-Luc, was generated. A fragment of the human adrenomedullin gene (–1866 to +8) was amplified using a 5' sense primer (GCCCCCTCGAGAGAAGTAAGAAAGAGAAGAAGCTGTGAT), a 3' antisense primer (CTTGTCAAGCTTCATCCCGGCTGCGAAGCGCA), and human leukocyte genome DNA as the template. The underlined nucleotides in the oligonucleotide sequences represent the XhoI and HindIII sites, respectively, which are not present in the adrenomedullin gene. Direct sequencing of amplified double-stranded PCR products was carried out in both directions by standard semiautomated methods using an ABI Prism 377 DNA Sequencer (PerkinElmer, Foster City, CA). PCR-amplified DNA fragments consisting of the human adrenomedullin gene and additional modified nucleotides were digested with XhoI and HindIII, and the resulting 1874-bp fragment spanning –1866 to +8 with sticky ends was subcloned into the XhoI/HindIII site of the pGL3-Basic Vector (Promega, Madison, WI). c-myc-, and max-expressing cDNAs (pSP-myc and pSP-max) were generously provided by Dr. R. N. Eisenman (Fred Hutchinson Cancer Research Center, Seattle, WA). The mouse adrenomedullin promoter construct, pGL2BmgAM5'-3 (24), containing a 3.0-kb sequence 5' to the mouse adrenomedullin ATG codon (generously provided by Dr. Elizabeth J. Taparowsky, Purdue University, West Lafayette, IN), was digested with KpnI/NdeI, blunt-ended, and religated to generate pmADM-Luc, which contains a –1439-bp upstream sequence. The pRL-TK vector expressing Renilla luciferase was purchased from Promega.

Cell culture
The rat fibroblast cell lines, TGR-1, HET15, HO15, HO16, and LACO3, which have been previously described, were gifts from Dr. John M. Sedivy (Brown University, Providence, RI) (20, 23, 25, 26, 27). Briefly, TGR-1 is a nontransformed diploid rat fibroblast cell line (25), and HET15 is an independent derivative of TGR-1 with one endogenous c-myc exon 2 knocked out by gene targeting using a neomycin-resistant sequence. LACO3 is a derivative of HET15, in which the c-myc transgene was stably introduced nonhomologously (26). HO15 and HO16 are derivatives of heterozygotes, selected after a second round of gene targeting using a hygromycin-resistant sequence to achieve c-myc-null phenotypes (20). Rat aortic endothelial cells were prepared from 15-wk-old male Wistar rats by collagenase and elastase digestion, as described previously (28, 29). The endothelial origin of the cultures was confirmed by the presence of factor VIII using an immunohistochemical method. A normal human skin diploid fibroblast cell line, Hs68, a human hepatic cancer cell line, PLC/PRF/5, and a human uterus tumor cell line, HeLa, were obtained from the Japanese Collection of Research Bioresources. Cells were cultured in DMEM in a 5% CO₂ atmosphere at 37 C supplemented with 10% fetal bovine serum, but HeLa cells were cultured in MEM and 10% calf serum was used for the panel of rat fibroblast cell lines.

Real-time quantitative RT-PCR
RNA was extracted from a single dish using RNAzol (GIBCO/BRL, Carlsbad, CA). First-strand cDNA from each mRNA was generated by reverse transcription using a First-Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech, Piscataway, NJ). Rat adrenomedullin and c-myc mRNAs were amplified using synthetic oligomers (adrenomedullin: forward: GTGGAATAAGTGGGCGCTAA, and complement: GCTGAGTGTGTCTGCTCCTG; c-myc exon 2: forward: TGCGACGAGGAAGAGAATTT, and complement: GCTTCTCGGAGACCAGTTTG; c-myc exon 3: forward: GTCCTCAAGAGGTGCCATGT, and complement: CTCGCCGTTTCCTCAGTAAG), detected, and quantified in real time using Light Cycler (Roche Diagnostics, Basel, Switzerland) as previously described (30, 31, 32). The amplification mixture contained 50 nM template cDNA and 500 µM primer DNA from a DNA amplification kit (DNA Master SYBR Green-I; Roche Diagnostics). For the measurement of adrenomedullin mRNA, denaturation was performed at 95 C, annealing at 62 C for 10 sec, and extension was performed at 72 C for 20 sec. The reaction produced a 411-bp PCR product that was confirmed by agarose gel electrophoresis. For the measurement of c-myc exon 2 and exon 3 mRNAs, denaturation was performed at 95 C, annealing at 57 C and 53 C for 5 sec, respectively, and extension was performed at 72 C for 20 sec. Quantification of the transcript number was expressed relative to serially diluted control samples.

Transient transfection
Transient transfections of phADM-Luc, pmADM-Luc, pSP-myc, and pSP-max were performed with a modified transferrin receptor-operated transfer with high efficiency as previously described (32, 33).

Transcription assay
phADM-Luc or pmADM-Luc (1 µg) was transiently cotransfected into subconfluent cells (96-well plates) with 1 µg of pSP-myc, pSP-max, or salmon testes DNA (Sigma-Aldrich Co., St. Louis, MO) and pRL-TK vector. Cells were then incubated in culture media containing 10% serum for 48 h, and luciferase activities were measured using the Dual Luciferase Reporter Assay System (Promega) in a single-tube assay format using MicroLumat^Plus (EG&G Berthold, Wildbad, Germany). The firefly luciferase activity of each sample was normalized to an internal reference standard of Renilla luciferase activity. All transfections were repeated two to four times in octets, with at least three different plasmid preparations; data from representative experiments are shown.

RIA
Cells grown in 10-cm dishes were incubated with serum-deprived media for 8 h, and 2 ml of supernatant was subjected to RIA. Adrenomedullin-like immunoreactivity was determined by an Adrenomedullin RIA kit (Phoenix Pharmaceuticals, Belmont, CA), according to the manufacturer’s instructions. The coefficients of variation of inter- and intraassays were less than 10%.

Statistical analysis
All results are expressed as means ± SEM. Statistical analysis was performed by using ANOVA for repeated measures. P < 0.05 was considered statistically significant.

	Results

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The plasmid, phADM-Luc, consists of the luciferase reporter gene behind the 1866-bp sequence spanning the 5'-flanking region of the human adrenomedullin gene (Fig. 1). The construct contains two noncanonical myc-binding sites upstream (CACGAG) and in intron 1 (CTCGTG). phADM-Luc was transiently cotransfected with c-myc cDNA and/or max cDNA into rat endothelial cells. c-myc cDNA caused significant stimulation of luciferase activity, whereas max cDNA had no appreciable effect (Fig. 2A). The reporter activity was significantly increased after cotransfection with c-myc and max (Fig. 2A), demonstrating the transactivation of phADM-Luc by c-myc. In contrast, the activity of pmADM-Luc containing an approximately similar length of mouse 5'-flanking sequence was suppressed by cotransfection with c-myc cDNA (Fig. 2B).

View larger version (9K): [in this window] [in a new window]   FIG. 1. Adrenomedullin reporter construct, phADM. The region between coordinates –1866 bp and +8 bp of the human adrenomedullin gene was placed upstream of luciferase in the promoterless plasmid, pGL3-basic. Two noncanonical c-myc binding sites, CACGAG and CTCGTG, identified by DNA sequencing, are indicated. Open boxes denote noncoding exons, and a hatched box indicates initial sequence encoding preproadrenomedullin. NF-IL6, nuclear factor for IL-6; AP, activator protein; SSRE, shear stress responsive element; CRE, cAMP-regulated enhancer element; GC, GC box; CAAT, CAAT box; TATA, TATA box.

View larger version (21K): [in this window] [in a new window]   FIG. 2. Effects of c-myc and/or max on the transcriptional activity of the adrenomedullin gene. A, Human adrenomedullin promoterreporter construct, phADM-Luc, and Renilla luciferase-expressing plasmid were cotransfected with salmon sperm DNA (control) or pSP-myc and/or pSP-max into rat endothelial cells. Firefly luciferase activity normalized to Renilla luciferase as an internal reference standard is presented as the relative mRNA level to that of control cells transfected with phADM-Luc, Renilla luciferase, and salmon sperm DNA. B, Mouse adrenomedullin promoter-reporter construct, pmADM-Luc, was cotransfected with salmon sperm DNA (control) or pSP-myc or pSP-max into rat endothelial cells, and relative reporter activity was determined. Each column with a bar shows the mean ± SEM. *, P < 0.05; **, P < 0.01, vs. control.

View larger version (15K): [in this window] [in a new window]   FIG. 3. phADM-Luc were cotransfected with either salmon sperm DNA (control) or pSP-myc into the human normal diploid fibroblast cell line, Hs68 (A), the human uterus cancer cell line, HeLa (B), or the human hepatoma-derived cell line, PLC/PRF/5 (C), and relative reporter activities were determined as in Fig. 2. *, P < 0.05; **, P < 0.01, vs. control.

View larger version (19K): [in this window] [in a new window]   FIG. 4. Quantification of c-myc exon 2, c-myc exon 3, and adrenomedullin mRNA levels in isogenic rat fibroblasts. c-myc exon 2 (A), c-myc exon 3 (B), and adrenomedullin (C) transcripts were quantified using LightCycler in a panel of fibroblasts genetically manipulated by targeted homologous recombination and stable transfection. The data are plotted as the relative mRNA levels of genetically manipulated cells to that of the c-myc diploid cells, TGR-1. Each column with a bar shows the mean ± SEM. *, P < 0.05; **, P < 0.01, vs. parental TGR-1. Bottom panels, Gel electrophoresis of RT-PCR products using primers amplifying either exon 2 (A) or exon 3 (B).

To determine whether transactivation of the adrenomedullin gene by c-myc results in markedly increased levels of adrenomedullin peptide synthesis, we measured the adrenomedullin-like immunoreactivity released from each cell line (Fig. 5). Compared with diploid parental cells, TGR-1, adrenomedullin release showed significant reductions in heterozygous and c-myc-null cells and a significant, but limited, increase in LACO3 cells (Fig. 5).

View larger version (27K): [in this window] [in a new window]   FIG. 5. Adrenomedullin-like immunoreactivity released from fibroblasts with differential c-myc expression. Cells were incubated in serum-free medium for 8 h, and the adrenomedullin released into the medium was measured by RIA. Each column with a bar shows the mean ± SEM. *, P < 0.05, vs. parental TGR-1.

View larger version (14K): [in this window] [in a new window]   FIG. 6. RNA degradation rate in fibroblasts expressing different c-myc levels. Total RNA was extracted from cells preincubated with actinomycin D for the indicated times, and adrenomedullin mRNA was quantified using real-time quantitative RT-PCR. The time courses of the changes of adrenomedullin mRNA levels in each cell line relative to diploid cells (TGR-1) are plotted against time.

	Discussion

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A number of genes have been proposed as c-myc targets, mainly using subtractive hybridization and differential display. However, because ectopic c-Myc influences cell-cycle progression, it is difficult to separate any direct effect of c-Myc from the indirect effects of changes in the cell cycle as overexpression approaches. A loss of function approach is critical for evaluating the potential of endogenous c-Myc in normal cellular physiology. In a study using c-myc-null cells, a limited number of the previously proposed c-myc targets proved to be regulated by c-myc (18). In the present study, we used isogenic fibroblast cell lines that were nontransformed and stably diploid and maintained the original morphological characteristics at the time of the experiments. Quantification of adrenomedullin mRNA revealed significant but limited suppression in heterozygous and null cells, demonstrating that c-myc is not essential for basal expression of the adrenomedullin gene. Other transcription factors might be involved in the steady-state expression of the adrenomedullin gene.

The heterozygous and homozygous c-myc cell lines used in this study are genetically stable and morphologically indistinguishable from wild types (20, 26). The observed effects are considered to be the direct consequences of c-myc knockout, and not secondary or tertiary effects, by the following lines of evidences. First, cell lines were obtained by targeted homologous recombination and confirmed to show no discernible additional genetic changes, such as nonhomologous integration of targeting module (20, 26). Second, two independent cell lines were confirmed to display the same phenotype at the initial (20) and second round of gene targeting experiments (26). Third, the phenotype was reversed by introducing a c-myc transgene (20). The heterozygous and homozygous cell lines are very stable in culture, and our quantification of the exon 2 and exon 3 c-myc sequences revealed that they maintained disrupted exon 2. A panel of cell lines, including the parental diploid cells, did not express N-myc or L-myc, which could compensate for the loss of c-myc, thus demonstrating that the homozygous knockout cell lines represent the c-myc-null phenotype (20, 25). Thus, we conclude that the adrenomedullin gene is induced by the c-myc transgene but is misregulated by the c-myc-null deletion.

Along with transcriptional regulation, RNA stability is also an important factor affecting gene expression levels. Each mRNA has a different half-life, and mRNAs that encode proteins produced in rapid response to stimuli tend to have short half-lives (36, 37). RNA sequences, such as the AU-rich element and iron-responsive element, are associated with RNA destabilization, whereas RNA stability can also be influenced by environmental factors such as hormones, stress, and cell differentiation. For example, TSH destabilizes RNA and down-regulates TSH-ß both transcriptionally and posttranscriptionally (37, 38), and hypoxia induces vascular endothelial growth factor through RNA stabilization (39). In contrast to these factors reported to influence RNA stability, transcriptional factors have rarely been described in relation to RNA decay. Down-regulation of c-myc during differentiation may be due, in part, to accelerated RNA turnover (40). In the present study, we have demonstrated enhanced adrenomedullin mRNA degradation in cells overexpressing c-myc, leading to a limited increase in adrenomedullin protein synthesis and subsequent cellular release, despite the adrenomedullin gene transactivation. Adrenomedullin release from the cells overexpressing c-myc was higher than the adrenomedullin release from the parental cells, but this does not appear to reflect the markedly elevated adrenomedullin mRNA levels in these cells. Thus, it is expected that c-Myc contributes to the synthesis and secretion of rat and human adrenomedullin upon its induction but may not exert an extended effect. Our finding that c-Myc destabilizes adrenomedullin RNA indicates the possibility that other transcriptional factors might also be involved in RNA turnover regulation. It is also tempting to speculate that c-Myc might destabilize RNA of other target genes, thus alleviating the effect of transactivation on their protein production.

The human adrenomedullin gene 5'-flanking region contains, in addition to the noncanonical c-myc binding site, TATA, CAAT, and GC boxes that are involved in the basal expression of the adrenomedullin gene (41). There are also multiple binding sites for activator protein-2 and a consensus sequence of the nuclear factor for the IL-6 binding site, suggesting roles for phospholipase C and protein kinase C in the regulation of adrenomedullin gene induction (Fig. 1). The presence of the cAMP-regulated enhancer element suggests a feedback mechanism by cAMP, whereas the shear stress responsive element and the interferon- responsive element imply involvement of physical and chemical stimuli in the transcriptional regulation. Our present results also demonstrate that, because rising adrenomedullin levels induce c-myc expression, elevated c-Myc protein acts to degrade adrenomedullin mRNA. Thus, regulation of adrenomedullin mRNA turnover by the c-myc gene, together with the transcriptional feedback mechanism at the cAMP-regulated enhancer element by cAMP produced by autocrine adrenomedullin stimulation, may constitute a built-in feedback signaling loop capable of finely regulating its own expression levels.

The intriguing question arises of whether there are any pathophysiological conditions that mirror these molecular findings. We previously demonstrated that adrenomedullin is abundantly expressed in injured vasculature and is actively involved in vascular remodeling at inflammation sites (42). Both adrenomedullin expression and neointimal thickening after balloon injury of the common carotid arteries were markedly suppressed by treatment with the adrenomedullin receptor antagonist, calcitonin gene-related peptide (8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37). In vascular smooth muscle cells at the site of injured vessels and in many cancer cells, abundantly expressed adrenomedullin acts as a potent growth-promoting factor (43). However, in injured vessels and other sites of inflammation, c-myc is induced by inflammatory cytokines as well as by physical and chemical stimuli, which may subsequently contribute to the degradation of adrenomedullin mRNA and the lessening of any overproduction of adrenomedullin. The fact that c-myc-induced expression of adrenomedullin is disrupted by high c-Myc levels can be viewed as a built-in safety mechanism for the maintenance of normal vascular pathology, such as in atherosclerosis, vascular remodeling, and tumor cell growth.

	Acknowledgments

 
We are grateful to Shinobu H. Yamaguchi and Hiroko Yasuda for technical assistance, to John M. Sedivy (Brown University, Providence, RI) for supplying the c-myc knockout cell lines, to R. N. Eisenman (Fred Hutchinson Cancer Research Center, Seattle, WA) for pSP-myc and pSP-max plasmids, and to Elizabeth J. Taparowsky (Purdue University, West Lafayette, IN) for pGL2BmgAM5'-3 plasmid.

	Footnotes

 
This work was supported in part by a grant-in-aid from the Ministry of Education, Science, Sports, Culture and Technology of Japan.

Received March 31, 2003.

Accepted for publication June 3, 2004.

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