Induction of Max by Adrenomedullin and Calcitonin Gene-Related Peptide Antagonizes Endothelial Apoptosis

Masayoshi Shichiri, Hiroki Kato, Masaru Doi, Fumiaki Marumo and Yukio Hirata

Division of Endocrinology and Metabolism Second Department of Internal Medicine Tokyo Medical and Dental University Tokyo 113-8519, Japan


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Adrenomedullin is a novel vasodilatory peptide originally isolated from pheochromocytoma. Recently, we found that adrenomedullin acts as an autocrine/paracrine apoptosis survival factor for rat endothelial cells. In the present study, we show that adrenomedullin induces the expression of Max, a heterodimeric partner of c-Myc, which may contribute to its ability to rescue endothelial cells from apoptosis. Max is a basic-helix-loop-helix-leucine zipper protein that forms heterodimers with its alternative partners, Mad and Mxi-1, to behave as an antagonist for Myc-Max heterodimer through competition for common DNA targets. The expression of Max is reported to be constitutive and more stable than c-Myc, and serum induces immediate c-Myc stimulation followed by modest Max up-regulation. In quiescent rat endothelial cells, adrenomedullin stimulated the expression of Max without affecting c-Myc. Quantitation with real-time quantitative PCR detected on the ABI Prism 7700 Sequence Detection System revealed that adrenomedullin and calcitonin gene-related peptide (CGRP), as well as serum, up-regulated Max mRNA levels and that down-regulation of Max mRNA after serum deprivation was prevented by adrenomedullin. Neither adrenomedullin nor CGRP affected c-Myc expression. Transfection of a Max-expressing plasmid into endothelial cells rescued the apoptosis induced by serum deprivation. Neutralization with anti-adrenomedullin antiserum or blockade with a CGRP receptor antagonist, CGRP(8–37), reduced Max mRNA levels in growing endothelial cells and enhanced apoptosis after serum starvation. Introduction of an antisense oligodeoxynucleotide against Max mRNA using transferrin receptor-operated transfer led to inhibition of both adrenomedullin-induced up-regulation of Max transcripts and its cell survival effect, whereas random, sense, or missense oligonucleotides were without effect. The negative regulation of E-box-driven transcription by adrenomedullin was demonstrated by using preproendothelin-1 promoter containing c-Myc-Max binding consensus sequence; the promoter activity of preproendothelin-1 was reduced by cotransfecting Max- and Mad-expressing plasmids as well as addition of adrenomedullin and CGRP. The present results demonstrate that adrenomedullin antagonizes serum deprivation-induced endothelial apoptosis by up-regulation of the max gene in an autocrine/paracrine manner.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Adrenomedullin is a potent vasorelaxant/hypotensive peptide with 52-amino acid residue originally isolated from human pheochromocytoma (1). Adrenomedullin has a conserved structure among mammals (2) and shows a partial homology with calcitonin gene-related peptide (CGRP) (1). Adrenomedullin is widely expressed in a variety of tissues, including vascular smooth muscle cells (VSMC) (3) and endothelial cells (4). Adrenomedullin receptors are functionally coupled to adenylate cyclase in VSMC and endothelial cells (5). We have recently found that adrenomedullin is not mitogenic in endothelial cells, but protects against apoptosis independent of cAMP activation (6), while it acts as a potent growth-promoting factor for quiescent VSMC via protein tyrosine kinase-mediated mitogen-activated protein kinase activation (7). Mitogenic activity of adrenomedullin is also reported in rat fibroblast cell lines (8), human tumor cell lines (9), and rat VSMC (7), whereas it inhibits proliferation of mesangial cells via cAMP-dependent mechanism (10).

Apoptosis, an intrinsic program to eliminate unwanted, aged, or damaged cells, is involved in the regulation of cell number in certain physiological and pathological conditions. Diverse stimuli, such as serum deprivation, radiation, chemotherapeutic agents, and antioxidants, induce apoptosis in many cell types (11, 12, 13), whereas growth factors and cytokines are known to prevent apoptosis triggered by such environmental signals (14). Forced expression of the cellular protooncogene c-myc combined with signals for entry into G0 has been shown to trigger apoptosis in fibroblasts (12) and in VSMC (15). Removal of serum growth factors from exponentially growing fibroblasts elicits a rapid loss of c-Myc at any position in the cell cycle (16). It has been suggested that c-Myc may integrate several signaling pathways since its induction is not solely dependent on any specific growth factors. Identification of the Myc dimerization partner Max has allowed a significant advance in our understanding of c-Myc function. Unlike c-Myc, Max expression is relatively stable throughout the cell cycle in hematopoietic cells and fibroblasts (17, 18), and the ability of c-Myc to promote proliferation, transformation, and apoptosis is believed to require dimerization with Max (19, 20). Max expression is reported to be growth regulated after a transient expression of c-Myc (21). Both Myc-Max heterodimers and Max-Max homodimers compete each other to bind to the E-box recognition site (CACGTG) and several related noncanonical sequences (22, 23, 24, 25). Myc-Max activates transcription to promote cell proliferation and apoptosis, while Max-Max, which lacks a transcriptional activation domain, represses it (26, 27). Max also forms a heterodimer with its alternative partners, Mad and Mxi-1, to behave as an antagonist for Myc-Max by competition for common DNA targets (28). Whether endogenous Max expression is affected by adrenomedullin to modulate apoptosis in endothelial cells with physiological c-Myc expression is yet unknown. In the present study, we have identified a unique function of adrenomedullin to up-regulate Max without inducing c-Myc expression, which prevented endothelial apoptosis triggered by serum deprivation.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Northern blot hybridization of total RNA from unstimulated rat endothelial cells revealed a distinct band corresponding to the size of Max mRNA (2.0 kb). Adrenomedullin (10-6 M) induced 2- to 3-fold increases in steady state Max mRNA levels in quiescent cells after 4–6 h (Fig. 1AGo), whereas it did not induce c-myc expression (data not shown). Western blot analysis using polyclonal anti-p21 Max antibody revealed that both adrenomedullin (10-6 M) and CGRP (10-6 M) increased p21 Max protein after 6 h (Fig. 1BGo), whereas neither forskolin (10-5 M) nor 8-bromo-cAMP (10-3 M) affected Max expression. Quantitation of Max mRNA transcripts using real-time quantitative PCR method revealed a time-dependent (2–6 h) increase in Max mRNA levels after treatment with adrenomedullin (10-7 M) (Fig. 2AGo), the effect of which was dose dependent (10-9–10-6 M) (Fig. 2BGo). Max mRNA expression was also up-regulated by 10% serum and CGRP (10-7 M) to the same extent as adrenomedullin (Fig. 2CGo). In contrast, c-Myc mRNA levels were unaffected by adrenomedullin or CGRP, but rapidly induced by 10% serum; adrenomedullin increased the ratio of Max to c-Myc in a time- (Fig. 2DGo) and dose-dependent (Fig. 2EGo) fashion, whereas the ratio after serum addition was comparable to that of control, which was in marked contrast to those after adrenomedullin and CGRP (Fig. 2FGo).



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Figure 1. Adrenomedullin Induces max Expression

A, Serum-deprived rat endothelial cells were treated with adrenomedullin (10-6 M) for 2–6 h. Total RNA (20 µg) was subjected to Northern blot analysis with max and GAPDH probes, respectively. Northern blotting was repeated four times with qualitatively similar results. B, Protein samples (15 µg) prepared from cell lysates after treatment with adrenomedullin, CGRP, forskolin, and 8-bromo cAMP for 6 h were subjected to Western blot analysis with anti-Max antibody (1:1000). An arrow shows the position of p21 Max protein. Western blotting was repeated three times with qualitatively similar results.

 


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Figure 2. Adrenomedullin Up-Regulates Max mRNA as Detected by Real-Time Quantitative PCR

Rat endothelial cells were rendered quiescent by incubating with medium containing 0.1% FBS for 24 h. Quiescent cells were stimulated with adrenomedullin (10-6 M) for the indicated time (panels A and D), the indicated concentrations of adrenomedullin for 6 h (panels B and E), or 10% serum, adrenomedullin (10-7 M), CGRP (10-7 M) for 6 h (panels C and F). Extracted total RNAs were subjected to quantitative PCR using the ABI Prism 7700 Sequence Detection System for the estimation of relative Max mRNA levels (A–C) and Max to c-Myc mRNA ratio (D–F) as described in Materials and Methods. Each column with bar shows mean ± SD (n = 4). *, P < 0.05; **, P < 0.01, treated vs. untreated or control cells.

 
Compared with quiescent cells, exponentially growing endothelial cells express about 4-fold greater Max mRNA levels, which decreased upon withdrawal of serum as a function of time (2–6 h) (Fig. 3AGo). The time-dependent down-regulation of Max mRNA expression by serum deprivation was antagonized by the addition of adrenomedullin (10-7 M). Serum withdrawal decreased c-Myc mRNA with or without adrenomedullin; thus adrenomedullin increased the ratio of Max to c-Myc (Fig. 3BGo). Addition of CGRP(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) (10-6 M), a CGRP/adrenomedullin receptor antagonist, and polyclonal antiadrenomedullin antiserum (1:100) resulted in a decrease in the steady state levels of Max mRNA (Fig. 4AGo) as well as the Max to c-Myc ratio (Fig. 4BGo); nonimmune serum (1:100) had no effect. Pretreatment with CGRP(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) and antiadrenomedullin antiserum enhanced apoptosis upon serum deprivation, while nonimmune serum was without effect (Fig. 4CGo). The results suggest that endogenous adrenomedullin contributes to maintain constitutive Max expression as an autocrine/paracrine factor to protect against apoptosis by serum deprivation.



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Figure 3. Adrenomedullin Antagonizes Serum Deprivation-Induced Max Down-Regulation

Exponentially growing rat endothelial cells were deprived of serum and incubated in the presence () or absence ({square}) of adrenomedullin (10-7 M) for the indicated time. Total RNAs were extracted and subjected to quantitative PCR as in Fig. 2Go for Max mRNA (A) and Max to c-Myc mRNA ratio (B). Each column with bar shows mean ± SD (n = 4). *, P < 0.05; **, P < 0.01, treated vs. untreated or control cells.

 


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Figure 4. Endogenous Adrenomedullin Induces Up-Regulation of Max mRNA and Protection from Apoptosis

A and B, Exponentially growing rat endothelial cells were incubated with or without CGRP(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 ) (10-7 M) or with either antiadrenomedullin antiserum or nonimmune serum (1:1000) for 72 h. Total RNAs were extracted and subjected to quantitative PCR for Max mRNA (A) and Max to c-Myc mRNA (B) ratio. Each column with bar shows mean ± SD (n = 4). C, Floating apoptotic cell number in a total cell population was counted 6 h after serum starvation after pretreatment with or without CGRP(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 ) (10-6 M) or with antiadrenomedullin antiserum or nonimmune serum (1:1,000) for 72 h. Each column with bar shows mean ± SD (n = 6). *, P < 0.05, treated vs. untreated or control cells.

 
We next determined whether overexpression of Max affects endothelial apoptosis. Transfection of a Max-expressing vector (pSP-max) into endothelial cells induced a preferential expression of Max p21 protein in a dose-dependent manner (20–80 µg/ml electroporation buffer) with a marginal p22 protein expression (Fig. 5AGo). After transfection with pSP-max plasmid, serum deprivation-induced apoptosis was inhibited as a function of doses used (Fig. 5BGo). The results suggest that rescue by Max from serum starvation-induced endothelial apoptosis is a Myc/Max-dependent process.



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Figure 5. Overexpression of Max Rescues Endothelial Cells from Serum Deprivation-Induced Apoptosis

A, Plasmid pSP-max in the indicated concentrations was electroporated into rat endothelial cells and cultured for 48 h. Protein samples (15 µg) were subjected to Western blot analysis with anti-Max antibody. Western blotting was repeated three times with qualitatively similar results. B, After electroporation and incubation for 48 h, cells were serum deprived for 6 h; all floating dead cells were collected and counted. Each bar represents mean ± SEM (n = 6); values were calculated to the percentage to the number of floating dead cells in the absence of pSP-max.

 
We reasoned that inhibition of Max down-regulation by adrenomedullin added upon serum deprivation protected against endothelial apoptosis. To block the adrenomedullin-induced Max mRNA expression, we treated the cells with antisense oligonucleotide against Max mRNAs by a transferrin receptor-operated transfer of liposome-encapsulated oligonucleotides. Quantitation of Max mRNA by a quantitative PCR method revealed that the adrenomedullin-induced up-regulation of Max mRNA was completely blocked by antisense oligonucleotide (0.2 µM), but not by sense, reverse, or missense oligonucleotides in the same concentrations (data not shown). Antisense oligonucleotide (0.2 µM) similarly antagonized cell survival effect of adrenomedullin, whereas sense, reverse, and missense oligonucleotides in the same concentrations were ineffective (Fig. 6Go).



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Figure 6. Max Antisense Oligonucleotides Abrogate Adreno-medullin (AM)-Induced Protection of Endothelial Apoptosis

Rat endothelial cells treated with or without 0.2 µM each of antisense, sense, reverse, and missense oligonucleotides against Max mRNA via transferrin receptor-mediated transfer were incubated in serum-free medium with or without 10-7 M adrenomedullin (AM) for 4 h as described in Materials and Methods; all floating dead cells were collected and counted. Data (n = 6) are plotted as in Fig. 5BGo. *, P < 0.05; **, P < 0.01, treated vs. untreated cells.

 
We examined whether adrenomedullin and CGRP in effect suppress E box-driven transcriptional activity using 1535 bp of 5'-flanking sequence of the human preproendothelin-1 gene containing noncannonical c-Myc-binding consensus (CACGTTG) (29) fused to the luciferase reporter gene (plasmid pPPET-luc). Cotransfection of pPPET-luc construct into growing rat endothelial cells with pSP-max and/or pSP-mad led to a marked suppression of the activity of pPPET-luc compared with that of control empty vector (Fig. 7AGo), suggesting negative regulation of preproendothelin-1 transcription by Max and Mad. Adrenomedullin and CGRP similarly and dose-dependently (10-10–10-6 M) suppressed the reporter gene expression, the effect of which was reversed by CGRP(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) (Fig. 7BGo).



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Figure 7. Preproendothelin-1 Promoter Activity Is Suppressed by Cotransfection with Max/Mad and by Adrenomedullin/CGRP

A, Plasmid pGL3-PPET1 was cotransfected with Renilla luciferase-expressing vector and with empty vector (pSP), Max-expressing plasmid (pSP-Max), and/or Mad-expressing plasmid (pSP-Mad) into rat endothelial cells and incubated for 48 h. B, Plasmid pGL3-PPET1 cotransfected with pRL-TK into rat endothelial cells was incubated for 24 h; the cells were then incubated with or without adrenomedullin (AM) and CGRP in concentrations indicated for 24 h. The ratio of Firefly to Renilla luciferase activity relative to that elicited by plasmid pGL3-PPET1 plus pSP are shown. Each bar represents mean ± SD (n = 8).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The present study demonstrates for the first time that a novel vasodilatory peptide, adrenomedullin, and CGRP induced Max gene expression in cultured rat endothelial cells without affecting its dimerization partner, c-Myc, as evidenced by the induction of Max mRNA and protein by Northern and Western blot analyses, respectively. This was further supported by quantitation with the real-time PCR method showing dose- and time-dependent up-regulation of Max transcripts by adrenomedullin. Our findings also revealed that serum deprivation induced down-regulation of Max mRNA, the effect of which was prevented by the addition of adrenomedullin, and that neutralization of endogenous adrenomedullin with antiadrenomedullin antiserum or by blockade of adrenomedullin/CGRP receptor with CGRP(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) reduced Max mRNA levels. Since rat endothelial cells used in the present study not only express the adrenomedullin gene and secrete mature peptide into the media, but contain its own receptors (6), our results are consistent with the notion that adrenomedullin produced by and released from endothelial cells could act in an autocrine/paracrine fashion to maintain constitutive Max expression.

The effect of adrenomedullin on Max expression is not restricted to endothelial cells. Adrenomedullin induces an immediate induction of c-Myc, but gradual and sustained expression of Max in rat VSMC (our unpublished observation). Such concomitant induction of Myc and Max by adrenomedullin may form Myc-Max heterodimers, possibly leading to transcription activation and mitogenic response. In contrast, adrenomedullin induces up-regulation of Max without affecting Myc levels in endothelial cells. Abundant Max protein in the absence of a corresponding increase in Myc protein may cause formation of Max-Mad or Max-Mxi1 heterodimers that are known to compete with Myc-Max heterodimer to its binding sites (Fig. 8Go). Thus, it is anticipated that up-regulation of Max protein may result in opposing physiological consequences depending upon the availability of Myc protein: in the presence of an abundant amount of Myc, Max may function as a transcription activator by forming Myc-Max heterodimer, whereas in the absence of sufficient Myc, Max may act as a transcription inhibitor for Myc-Max.



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Figure 8. A Proposed Mechanism of Antiapoptosis Induced by Adrenomedullin (AM) and CGRP in Rat Endothelial Cells

Up-regulation of Max protein in the absence of c-Myc induction may result in formation of Max-Mad or Max-Mxi heterodimers, which compete with Myc-Max to E-box as a transcription inhibitor. Boxes indicate genes; Myc, Max, Mad, and Mxi indicate the encoded gene products. An arrowhead with a straight lineindicates a positive effect. A bold solid arrow indicates a promoter action. CACGTG, a cannonical binding site for Myc-Max, Max-Mad, or Max-Mxi.

 
Transcriptional inhibition by adrenomedullin and CGRP was further confirmed by the experiments using preproendothelin-1 promoter-luciferase construct, which contains a noncannonical c-Myc-Max binding site (29). Cotransfection of a Max- and/or Mad-expressing plasmid suppressed the promoter activity, suggesting a transcription inhibition by antagonizing c-Myc-Max. Addition of either adrenomedullin or CGRP to the cells transfected with preproendothelin-1 promoter-luciferase construct similarly and dose-dependently inhibited the promoter activity, the effect of which was reversed by a CGRP receptor antagonist. These results suggest that both adrenomedullin and CGRP reduced E-box-driven transcriptional activity via common receptor.

Myc-induced apoptosis is dependent upon the level at which it is expressed, and induction of apoptosis by c-Myc requires association with Max, suggesting that c-Myc drives apoptosis through a transcriptional mechanism (20, 30, 31). Myc-Max dimerization is also a prerequisite for cell cycle progression in fibroblasts (20). The apparently paradoxical functions have been demonstrated to be antagonized by overexpressing Max, whose homodimers or heterodimers with Mad compete with Myc-Max heterodimers for its specific DNA target sites (28). Recent studies report apparently contradictory apoptotic responses to Max overexpression experiments using different experimental designs or cell types (32, 33), implying differential effects on apoptotic events depending on Max expression levels and availability of Myc protein. In a nontransformed fibroblast cell line selected from Rat-1 cells where c-myc is diploid and constitutive c-Myc expression level is very low without N- or L-Myc expression (34), overexpression of Max also resulted in a marked suppression of apoptosis (our unpublished observation). It is presently unknown how Myc or Max is involved in endothelial apoptosis. The present study revealed that cell death induced by serum deprivation was rescued by overexpressing Max, suggesting that Myc protein, possibly by dimerizing with constitutive Max, plays an important role in the mechanism of apoptosis in endothelial cells (Fig. 8Go).

The significance of Max function was further tested in vitro by using an efficient transferrin receptor-operated transfer of oligonucleotides that clearly showed that the adrenomedullin-induced up-regulation of Max mRNA was completely blocked by antisense oligonucleotide, but not by either sense, reverse, or missense oligonucleotides. Concomitantly, suppression of Max up-regulation after selective transfer of antisense oligonucleotide led to abrogation of adrenomedullin-induced protection from endothelial apoptosis. Furthermore, both neutralization of adrenomedullin with antiadrenomedullin antibody and blockade of CGRP/adrenomedullin receptor with CGRP(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) caused down-regulation of Max mRNA and increased apoptotic events upon serum deprivation. Taken together, the results are consistent with the notion that Max plays a pivotal role in the mechanism of survival effect by adrenomedullin from endothelial apoptosis.

In this study, adrenomedullin and CGRP similarly up-regulated Max without affecting c-Myc expression and suppressed E-box-driven transcriptional activity, the concentrations of which appear to be comparable. These data suggest that adrenomedullin and CGRP share the same and/or very similar, if not identical, receptors. Notably, it has recently been shown that calcitonin-receptor-like receptor functions as either CGRP receptor or adrenomedullin receptor depending upon whether receptor activity-modifying protein (RAMP) 1 or RAMP2 is simultaneously expressed, respectively (35). It remains to be clarified as to which RAMP isoforms are predominantly expressed by rat endothelial cells.

Adrenomedullin was initially identified with its ability to stimulate cAMP formation in platelets, and cAMP has been suggested as a second messenger for its observed vasorelaxation effects (1). However, we reported previously that protection of endothelial apoptosis by adrenomedullin is not mediated via cAMP, because cAMP-elevating agonists (forskolin, PGI2) did not inhibit apoptosis, and a cAMP antagonist (Rp-cAMP) failed to block adrenomedullin-induced antiapoptotic effect (6). The present study revealed that both forskolin, an activator of adenylate cyclase, and 8-bromo-cAMP failed to induce Max protein expression, suggesting that up-regulation of Max is independent of cAMP. We have recently shown that adrenomedullin has a growth-promoting effect via tyrosine kinase-mediated mitogen-activated protein kinase activation in rat VSMC (7). Furthermore, it has been reported that adrenomedullin stimulates phospholipase C to generate inositol-1,4,5-trisphosphate (IP3), which mobilized intracellular Ca2+, thereby leading to nitric oxide production in bovine endothelial cells (36), although adrenomedullin had no effects on IP3 generation or intracellular Ca2+ concentrations in our rat endothelial cells (6). Thus, there appear to exist multiple signal transduction pathways of adrenomedullin in addition to the cAMP-dependent pathway. The exact signal transduction of adrenomedullin linked to Max induction remains to be determined.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture
Rat endothelial cells were prepared from 15-week-old male Wistar rat aorta (Saitama Experimental Animals Supply Co., Saitama, Japan) by collagenase and elastase digestion, as described previously (6, 37). The endothelial origin of the cultures was confirmed by the cobblestone appearance and the presence of Factor VIII by immunohistochemical method. Cells (passages 5–10) were cultured in DMEM in a 5% CO2 atmosphere at 37 C, supplemented with 10% FBS.

Reagents
Synthetic rat adrenomedullin, rat CGRP, and human CGRP(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) were purchased from Peptide Institute (Osaka, Japan), DMEM from Life Technologies, Inc. (Rockville, MD), FBS from HyClone Laboratories, Inc. (Logan, UT), forskolin from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), and 8-bromo-cAMP from Sigma Chemical Co. (St. Louis, MO). All other reagents were of analytical grade. pSP-Max and pSP-Mad plasmids were kindly supplied by Dr. R. N. Eisenman, Fred Hutchinson Cancer Research Center (Seattle, WA).

Detection of Apoptosis
We described previously that primary rat endothelial cells undergoing apoptosis are detached from culture plates after serum deprivation and that floating apoptotic cell number increases as a function of time, reaching a significant fraction (~40%) within 24 h (6, 37). This was proven by 1) demonstration of nucleosomal ladders of extracted fragmented DNA from total culture using a highly efficient NP-40 lysis method that efficiently eliminates intact chromatin; 2) the presence of hypodiploid cells almost exclusively in the floating fraction of endothelial culture stained with propidium iodide by flow cytometric analysis; 3) terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method; 4) immunohistochemical detection using antibody against single-stranded DNA; and 5) morphological features characteristic of apoptosis. Determination of floating and adherent fractions of endothelial culture by FACS Calibur flow cytometer (Becton Dickinson, San Jose, CA) confirmed our previous results that collections of floating fraction by two washes with PBS and adherent fraction by trypsinization clearly separate apoptotic and viable cells, respectively (6, 37). Therefore, the percentages of apoptotic cells were calculated by counting floating apoptotic and trypsinized viable cell number using a Sysmex CDA-500 Autoanalyzer (Toa Medical Electronics, Kobe, Japan). In brief, rat endothelial cells were plated in 24-well dishes in serum-containing medium and incubated for 24 h. The cells were extensively washed with PBS, replaced with serum-free DMEM, and incubated with or without the indicated reagent. After 4 h, all floating cells were collected after two washes with PBS as described previously (6, 37, 38). All adherent cells were also collected after trypsinization for a quantitative analysis of total apoptotic events in a given cell population. For determination of the involvement of endogenous adrenomedullin, cells were plated in 24-well dishes in serum-containing medium and incubated with or without CGRP(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), nonimmune control rabbit serum, or rabbit polyclonal antiadrenomedullin antiserum (39) for 72 h. The medium was then changed to serum-free DMEM, and all floating and adherent cells were counted after 4 h.

Northern Blot Analysis
RNA was extracted from rat endothelial cells by the guanidinium thiocyanate method as described (40). Total RNA (20 µg per lane) was electrophoresed on formaldehyde-agarose gels. cDNA probes for rat adrenomedullin, max, c-myc, and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were labeled with {alpha}-[32P]dCTP using the random-priming method. Signals were quantitated using a BAS2000 Imaging Analyzer (Fuji Photo Film Co., Ltd.). All values were corrected against the GAPDH internal control. To confirm reproducibility of the Northern hybridization detection, each RNA sample was assayed independently on four occasions.

Real-Time Quantitative PCR
Real-time quantitative PCR method was used to detect accurately the changes of max gene copies. The cycle at which the amplification plot crosses the threshold (CT) is known to accurately reflect relative mRNA values (41, 42). Total RNA was harvested from cells treated with or without adrenomedullin or indicated reagents. Rat max and c-myc mRNAs were amplified (max: forward primer 5'-ACGA-TGACATCGAGGTGGAGAG-3' and reverse primer 5'-GCA-TTATGGTGAGCCCGTTT-3', c-myc: forward primer 5'-AAAGGCCCCCAAGGTAGTTATC-3' and reverse primer 5'-GTGCTCATCTGCTTGAACGG-3'), detected, and quantitated in real time using the ABI Prism 7700 Sequence Detection System (PE Applied Biosystems, Foster City, CA) as described previously (41, 42). The TaqMan probes for max and c-myc were 5'-ACCGAGGTTTCAATCTGCGGCTGA-3' and 5'-TCAAAAAAGCCACCGCCTACATCCTG-3', respectively. The amplification mixture contained 5 nM of template DNA and 50 µM of primer DNA in 50 mM salt and 1 mM Mg2+. A three-step PCR was performed for 35 cycles. Denaturation was done at 94 C for 20 sec, annealing at 55 C for 20 sec, and extension at 72 C for 30 sec. The reaction produced a 79-bp PCR product for max and 70-bp product for c-myc. The PCR cycle at which the amplification plot crosses a threshold of 10 SD above the baseline was defined as a CT value. In comparison to control sample without RNA, which did not show any increase in normalized reporter signal (Rn), addition of RNA from rat endothelial cells resulted in PCR amplification reaching the threshold value. Relative mRNA level was calculated based on the assumption that CT values increase by approximately 1 for each 2-fold dilution (41, 42). To determine precision of the assay, mRNA from endothelial cells was reverse transcribed and amplified on three separate days. The mean CT values ranged from 22.34 to 22.59, and an intraassay precision from 0.17% to 0.42% coefficient of variation. The interassay precision of amplification for the 3 days was 3.2%.

Western Blot Analysis
Confluent cells on 10-cm dishes were harvested in PBS on ice, pelleted and resuspended in 200 µl of ice-cold 62.5 mM Tris (pH 6.8), 10% (wt/vol) glycerol, and 1 mM phenylmethylsulfonyl fluoride. Fifteen microliters of 20% SDS were added, vortexed, and immediately boiled for 5 min. Protein was quantitated using the Micro BCA Protein Assay Kit (Pierce Chemical Co., Rockford, IL). Western blotting analysis was performed as described (43, 44). SDS polyacrylamide gels were transferred onto nitrocellulose, which was blocked with Blotto (5% dried nonfat milk in PBS), and incubated with affinity-purified rabbit polyclonal antibody raised against the carboxy terminus of Max p21 (C-17, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), followed by horseradish peroxidase-conjugated antirabbit IgG antiserum (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Signals were visualized using the ECL Chemiluminescence System (Amersham Pharmacia Biotech, Arlington Heights, IL). Membranes were stained with Pelikan Fount India Ink to verify electrotransfer uniformity (45).

Overexpression of max Gene
For experiments overexpressing max gene, pSP-Max plasmid (20–80 µg/ml electroporation buffer) was electroporated into subconfluent cells cultured in 10-cm dishes using a Gene Pulser apparatus (Bio-Rad Laboratories, Inc., Richmond, CA), as described previously (29), and plated in 24-well dishes. After 48 h incubation, the cells are deprived of serum and incubated for 4 h; floating and adherent cells were counted. pSVßGAL plasmid DNA (5 µg) was included in all transfections to assure almost equal electroporation efficiency. Salmon sperm DNA was used as carrier to adjust the total DNA content of each electroporation sample to 85 µg/ml.

Transfer of Oligodeoxynucleotides for Max mRNA
An antisense oligodeoxyribonucleotide, a phosphorothioate-protected 15-mer directed against the initiation of translation site of rat Max mRNA (5'-ATCGTTATCGCTCAT-3'), sense (5'-ATGAGCGATAACGAT-3'), missense (5'-ATCGTGA-TCTCTCAT-3'), and reverse (5'-TAGCAATAGCGAGTA-3') oligonucleotides was synthesized and introduced into endothelial cells using transferrin receptor-mediated transfer (46). In brief, 3 µl of Lipofectin (1 mg/ml, Life Technologies, Inc.) was added to 100 µl HEPES-buffered saline (HBS, 20 mM HEPES, pH 7.4, and 100 mM NaCl) containing 16 µg human holo-transferrin (Sigma Chemical Co.), incubated for 20 min at room temperature, mixed with 100 µl HBS containing various amount of oligonucleotide complexes, and further incubated for 15 min. The mixture was then overlaid on cells that had been covered with 300 µl of serum-free DMEM and incubated for 4 h with or without adrenomedullin. To determine the transfection efficiency, fluorescein isothiocyanate-conjugated oligonucleotides were introduced into rat endothelial cells in the same manner as indicated above. Regardless of the oligonucleotide concentration used (0.1–10 µM), nearly 100% of total cells were transfected, and the intensity of cellular fluorescence correlated well with the oligonucleotide concentration used. In a control experiment, the antisense oligomer (0.2–1 µM) markedly reduced Max mRNA levels; 0.5–1 µM of the antisense reduced the Max mRNA levels below the baseline value, and 0.2 µM of antisense blocked the rise of Max mRNA by adrenomedullin, whereas sense, missense, and reverse in the same concentrations were without effect. Therefore, a 0.2 µM concentration was used in oligonucleotide transfer experiment. Therefore, cells plated in 24-well dishes and incubated for 24 h were treated with 0.2 µM each of oligonucleotides via transferrin receptor-operated transfer; floating and adherent cells were counted after 4 h.

Transcriptional Activity of Preproendothelin-1 Promoter
A PCR-generated, XhoI- and HindIII-tailed, DNA fragment (-1535 to +86 bp) of sequence 5' to the human preproendothelin-1 gene containing noncannonical c-Myc-binding consensus was cloned into the XhoI and HindIII site of pGL3 basic (Promega Corp., Madison, WI) to create pGL3-PPET1. Cells plated in 96-well plates were transiently cotransfected with pGL3-PPET1 and pRL-TK vector (1 µg each/well) (Promega Corp.) expressing Renilla luciferase as an internal control using synthetic cationic lipid, (+)-N,N [bis (2-hydroxyethyl)-N-methyl-N-[2,3-di(tetradecanoyloxy)propyl] ammonium iodide (Promega Corp.) according to the manufacturer’s suggestion. Firefly and Renilla luciferase activities were measured using the Dual Luciferase Reporter Assay System (Promega Corp.) in a single-tube assay format using MicroLumatPlus (EG&G Berthold, Wildbad, Germany).


    ACKNOWLEDGMENTS
 
The authors gratefully acknowledge R. N. Eisenman, Ph.D., Fred Hutchinson Cancer Research Center (Seattle, WA) for pSP-Max and pSP-Mad plasmids, Shinobu Yamaguchi for her expert technical assistance, and Nakanobu Hayashi, M.D., Omgen Inc., for allowing us to use ABI 7700 TaqMan Sequence Detector and for help in quantitation of Max and c-Myc mRNA.


    FOOTNOTES
 
Address requests for reprints to: Masayoshi Shichiri, M.D., Second Department of Internal Medicine, Tokyo Medical and Dental University, 1–5-45, Yushima, Bunkyo-ku, Tokyo 113-8519, Japan.

This work was supported in part by the Ministry of Education, Science and Culture, Japan, and by the Ministry of Health and Welfare, Japan.

Received for publication December 14, 1998. Revision received April 28, 1999. Accepted for publication May 3, 1999.


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 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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