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Endocrinology Vol. 138, No. 11 4584-4590
Copyright © 1997 by The Endocrine Society

ARTICLES

Biphasic Regulation of the Preproendothelin-1 Gene by c-myc1

Masayoshi Shichiri, Susumu Adachi, John M. Sedivy, Fumiaki Marumo and Yukio Hirata

Endocrine-Hypertension Division, Second Department of Internal Medicine (M.S., S.A., F.M., Y.H.), Tokyo Medical and Dental University, Tokyo 113, Japan; and the Department of Molecular Biology, Cell Biology and Biochemistry (J.M.S.), Brown University, Providence, Rhode Island 02912

Address all correspondence and requests for reprints to: Dr. Masayoshi Shichiri, Second Department of Internal Medicine, Tokyo Medical and Dental University, 1–5-45 Yushima, Bunkyo-ku, Tokyo 113, Japan. E-mail: mshichiri.med2{at}med.tmd.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Endothelin-1 (ET-1), a potent vasoconstrictive/mitogenic peptide originally isolated from vascular endothelium, stimulates the expression of immediate early response genes such as c-myc. The c-myc protooncogene participates in regulating the cascade of events that follow mitogenic stimulation of quiescent cells. Using a panel of isogenic fibroblast cell lines with differential c-myc expression levels (obtained by disrupting one c-myc gene copy with targeted homologous recombination and subsequently stably transfecting the heterozygous cells with an exogenous c-myc transgene), we demonstrate that c-Myc protein regulates ET-1 gene transcription in a biphasic fashion: as an activator at low concentrations and as a repressor at high concentrations. Using rat endothelial cells treated with antisense c-myc oligodeoxynucleotides, we also show that c-myc regulates ET-1 synthesis and secretion in a biphasic manner. The present report, therefore, demonstrates the existence of a signal transduction pathway that regulates the synthesis and secretion of ET-1 via the immediate early transcription factor, c-Myc.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ENDOTHELIN-1 (ET-1) is an endothelium-derived 21-residue peptide that mediates a variety of physiological functions, such as vasoconstriction and cell proliferation (1, 2). ET-1 and its isopeptides, ET-2 and ET-3, were subsequently found to play diverse physiological roles, mediated by two distinct subtypes of G protein-coupled receptors, designated ETA and ETB, that are expressed in a wide variety of tissues (3, 4). The synthesis and secretion of ET-1 are tightly regulated in response to various humoral and mechanical stimuli (5). Reporter gene transfection experiments revealed the importance of both a GATA-2 site and an AP-1 site for promoter function of the human ET-1 gene in endothelial cells (6, 7, 8). ET-1 is also known to be a potent mitogen; it stimulates the proliferation of many vascular and nonvascular cells (9, 10), it induces the expression of immediate early response genes, such as c-myc (10), and in some malignant cell lines, it functions as an autocrine/paracrine growth factor (11). Disruption of the ET-1 gene in mice results in fatal craniofacial malformations of tissues derived from the first branchial arch, indicating its essential role in the development of neural crest-derived tissues (12).

The c-myc protooncogene is an immediate early growth response gene and participates in regulating the cascade of events that follows mitogenic stimulation of quiescent cells. Accumulating evidence suggests that c-Myc regulates proliferation, mitogenesis, differentiation, and programed cell death (13, 14). c-Myc is a transcription factor with a basic region-helix-loop-helix-leucine zipper DNA-binding domain, acts as a heterodimer with a partner designated Max (15, 16), and binds to the E-box recognition site CACGTG and several related noncanonical sequences (16, 17, 18, 19). As other transcription factors also bind to the E-box motif, identification of physiologically relevant c-Myc target genes has been difficult. Several genes have been found to be activated by c-Myc via an E-box site: plasminogen activator inhibitor-1 (20), {alpha}-prothymosin (21), ECA 39 (22), p53 (23), ornithine decarboxylase (24), and dihydrofolate reductase (25). Recently, it has been shown that cdc25A is a transcriptional target of c-Myc and mediates c-Myc-induced apoptosis (26). However, the c-Myc targets responsible for mediating its growth regulatory functions remain largely unknown. c-Myc can also repress transcription through the initiator element (27), and in this fashion is thought to participate in the biphasic regulation of the adenovirus-2 major late promoter (28).

Our understanding of c-Myc function mostly derives from studies of cells overexpressing the c-myc gene. Using targeted homologous recombination, we previously disrupted one c-myc gene copy in a diploid fibroblast cell line to investigate the consequences of reduced c-myc expression (29). Heterozygous c-myc cells are genetically stable and free from additional genetic changes and express approximately 50% of normal diploid c-Myc levels. This subtle perturbation of c-myc expression resulted in slower growth rates, lengthening of the G0 to S cell cycle transition, and modulation of cyclin E expression (30). In the present study, we show that c-Myc regulates transcription of the ET-1 gene in a biphasic fashion. We also demonstrate that introduction of c-myc antisense oligonucleotides into rat endothelial cells alters the levels of endogenous ET-1 expression. We propose that ET-1 and c-Myc constitute a positive/negative feedback signaling loop capable of finely regulating its own expression and, consequently, cell proliferation and apoptosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and cell culture
The rat fibroblast cell lines TGR-1, HET15, HET16, LACO3, and LACO16 are all derivatives of the Rat-1 cell line, and have been previously described (29, 30, 31). TGR-1 is a nontransformed diploid rat fibroblast cell line (31), HET15 and HET16 are two independent TGR-1 derivatives with one endogenous c-myc copy knocked out by gene targeting, and LACO3 and LACO16 are derivatives of HET15 and HET16, respectively, in which c-myc expression was restored by transfection. HET15 and HET16 express 50% of normal diploid c-Myc levels, and LACO3 and LACO16 overexpress c-Myc approximately 4-fold relative to the diploid condition (29). Rat aortic endothelial cells were prepared from 15-week-old male Wistar rats by collagenase and elastase digestion, as described previously (32, 33). The endothelial origin of the cultures was confirmed by the presence of factor VIII using the immunohistochemical method. All cells were cultured in DMEM in a 5% CO2 atmosphere at 37 C supplemented with 10% calf serum (fibroblasts) or 10% FBS (endothelial cells).

Reagents
Phosphorothioate oligodeoxynucleotides were synthesized at Sawaday Technology (Tokyo, Japan). An oligodeoxyribonucleotide complementary to the first five codons of rat c-myc messenger RNA (mRNA; rat c-myc antisense, 5'-CACGTTGAGGGGCAT) and its reverse (c-myc reverse, 5'-GTGCAACTCCCCGTA), missense (c-myc missense, 5'-CACGTGGAGTGGCAT), and sense (c-myc sense, 5'-ATGCCCCTCAACGTG) controls were introduced into rat endothelial cells by the Lipofection (Life Technologies, Grand Island, NY) method (34). In brief, oligonucleotides were dissolved in serum-free DMEM medium and mixed with Lipofectin reagent, and the liposome-oligonucleotide complexes were incubated for 20 min at room temperature and overlaid on cells that had been extensively washed with serum-free DMEM. In a control experiment, 2.5 µM of the antisense oligomer reduced c-Myc mRNA levels measured by ribonuclease protection (29) by 2-fold. To determine the transfection efficiency, FITC-conjugated oligonucleotides were introduced into rat endothelial cells in the same manner as indicated above. The transfection efficiency by the lipofection method, regardless of the oligonucleotide concentration used (0.1–10 µM), was approximately 50% of the total cells, and the intensity of cellular fluorescence correlated well with the oligonucleotide concentration used.

Plasmids
Human ET-1 promoter sequences were cloned by PCR. The primers used were as follows: sense strand, 5'-CGAAGCTTCTAGTAAAACATCAGCCCCT-3', spanning the -875- to -855-bp fragment with the addition of a HindIII restriction site at the 5'-ends; and antisense strand, 5'-ACGCGTCCCAAGGAGTCTAGAGTTTCG-3'. The PCR product corresponding to the full-length product was excised, digested with PstI and HindIII, cloned into PstI- and HindIII-cut pCAT-Basic vector (Promega, Madison, WI), and sequenced (plasmid pET1{Delta}875). Plasmid pET1{Delta}875 contains the -875- to +51-bp fragment of the human ET-1 gene upstream of the chloramphenicol acetyltransferase (CAT) gene. Plasmids pET1{Delta}642, pET1{Delta}372, and pET1{Delta}179 were generated by digesting the 925-bp PstI-HindIII fragment with SacI, DraI, and AcyI, respectively, and subsequently cloning the truncated fragments into the same cloning site of pCAT-Basic. Plasmid pET1{Delta}20 was constructed by deleting an 855-bp NaeI-HindIII fragment of pET1{Delta}875. The c-Myc expression plasmid, pSPT-Myc, originally developed by Dr. N. Nomura was obtained from the Japanese Cancer Research Resources Bank. pSVßGAL was purchased from Promega. All recombinant DNA manipulations were performed using standard procedures.

CAT assays
Rat fibroblast cell lines and endothelial cells were electroporated as described previously (31). Electroporation was carried out in a Gene Pulser apparatus (Bio-Rad, Richmond, CA) using a 0.4-cm gap cuvette at the settings of 210 V and 960 µF. The amounts of individual plasmid DNAs in the electroporation samples are indicated in the text. To assess the electroporation efficiency, pSVßGAL plasmid DNA (5 µg) was included in all transfections. Salmon sperm DNA was used as a carrier to adjust the total DNA content of each electroporation sample to 250 µg/ml. Cell extracts, prepared 48 h after transfection, were subjected to measurements of protein concentrations with the Bio-Rad DC kit and of CAT activity (35). Thin layer chromatographs were quantitated using a BAS2000 Imaging Analyzer (Fuji Photo Film, Tokyo, Japan). CAT activities were expressed as the rate of substrate conversion, which was normalized to the ß-galactosidase cotransfection control value. To confirm reproducibility of the experiments, all transfection experiments were repeated on at least three occasions.

RIA of ET-1
ET-1-like immunoreactivity was determined by double antibody RIA, as previously described (11, 36): the antibody used was directed toward the C-terminal Trp21 residue of ET-1 without any cross-reactivity with big ET-1. Rat aortic endothelial cells under serum-free conditions secreted ET-1-like immunoreactivity as a function of time (9.65 ± 2.27 fmol/24 h·106 cells; n = 4).

Northern hybridization analysis
RNA was extracted from rat endothelial cells by the guanidinium thiocyanate method, as previously described (11). Total RNA (15 µg) was electrophoresed on formaldehyde-agarose gels, and transferred to MagnaGraph nylon membranes (Micron Separations, Westborough, MA). Complementary DNA probes for rat ET-1 and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) genes were labeled with [{alpha}-32P]deoxy-CTP using the random priming method. After UV cross-linking, membranes were hybridized at 42 C in the presence of 50% formamide. Washing was performed with 0.1 x SSPE (15 mM NaCl, 1 mM NaH2PO4, and 0.1 mM EDTA) and 0.5% SDS at 37 C for 15 min, and signals were quantitated using a BAS2000 Imaging Analyzer (Fuji Photo Film). All values were corrected against the GAPDH internal control. To confirm reproducibility, Northern hybridization was performed on four independent occasions.

Statistical analysis
Data are expressed as the mean ± SEM. Statistical analysis were performed using ANOVA for repeated measures.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Biphasic regulation of ET-1 gene expression by c-Myc
To determine whether c-Myc influences ET-1 expression, 875 bp of the 5'-flanking sequence of the human ET-1 gene were fused to the CAT reporter gene (plasmid pET1{Delta}875; Fig. 1Go). The construct was transiently electroporated into a panel of isogenic cell lines with different c-myc expression levels. Compared with TGR-1 cells, CAT reporter expression was significantly (P < 0.01) attenuated in two heterozygous cells, HET15 (2.2 ± 0.8%) and HET16 (13.5 ± 0.7%), as well as in LACO3 (7.3 ± 0.7%) and LACO16 (0.4 ± 0.4%), which overexpress the c-myc gene (Fig. 2Go). These data suggest that altered expression of c-myc genes results in a marked suppression of ET-1 gene expression.



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  		Figure 1. ET-1 reporter construct and deletion derivatives. The region between coordinates -875 bp and +51 bp (relative to the start of transcription) of the human ET-1 gene was placed upstream of CAT in the promoterless plasmid pCAT-Basic, and the series of deletions shown was constructed. A potential c-Myc binding site, CATGTG (19), identified by DNA sequencing, is indicated. Also shown are binding sites for known transcription factors: NF-1 (position -292/-277), TATC (position -135/-132), AP-1/Jun (position -108/-104), and CACAAT (position -98/-94). A canonical TATAAA sequence is located at position -31/-26. The bent arrow represents the start of transcription.

 


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  		Figure 2. Activity of ET-1 promoter in a panel of fibroblast cell lines with differential c-Myc expression levels. Plasmid pET{Delta}875 was cotransfected with plasmid pSVßGAL into cell lines TGR-1, HET15, HET16, LACO3, and LACO16; equal amounts of extract (15 µg protein) were used for each lane (see Materials and Methods). CAT activities, expressed as the rate of substrate conversion and normalized to the ß-galactosidase cotransfection controls, are displayed relative to those by plasmid pET{Delta}875 transfected into TGR-1 cells. Each column with a bar shows the mean ± SEM (n = 3). *, P < 0.01, TGR-1 vs. genetically manipulated cells.

 
To gain insight into this phenomenon, we examined the ability of a c-Myc expression vector (pSPT-Myc) to transactivate the pET1{Delta}875 construct in transient cotransfection assays of HET15 and HET16 cells. In HET15 cells, transfection with a lower concentration of pSPT-Myc DNA (40 µg/ml) resulted in a marked stimulation (25- to 30-fold), whereas reporter gene expression was repressed to basal levels at higher concentration (80 µg/ml; Fig. 3Go). Similar data were obtained with HET16 cells (data not shown). Taken together, these data indicate that c-Myc is a strong transactivator of the ET-1 promoter at physiological (diploid) c-Myc expression levels, but that elevated c-Myc expression levels have a suppressive effect.



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  		Figure 3. Cotransfection of ET-1 promoter deletion constructs with a c-Myc expression vector. Equal amounts (40 µg) of the indicated ET-1 reporter constructs were cotransfected with increasing amounts of pSPT-Myc plasmid into HET15 cells. CAT activities are displayed relative to the activity elicited by plasmid pET{Delta}875 in the absence of the c-Myc vector. The experiment was performed three times with consistent results; a representative experiment is shown.

 
Deletion analysis of the human ET-1 gene promoter
To localize the regions responsible for c-Myc regulation of ET-1 gene expression, a series of promoter deletion mutants was constructed (Fig. 1Go). In transient transfections of TGR-1 cells, truncation of the ET-1 promoter at -642 bp (pET1{Delta}642) elicited lower CAT activity (20.0 ± 1.5%) than the full-length promoter (pET1{Delta}875), whereas truncation at position -372 bp (pET1{Delta}372) enhanced CAT activity (452.7 ± 23.8%) relative to that produced by pET1{Delta}875 (Fig. 4Go). Further deletions to -179 and -20 bp (pET1{Delta}179 and pET1{Delta}20) resulted in only low basal levels of expression (9.9 ± 0.0% and 32.0 ± 1.5%, respectively). In HET15 cells, however, the overall level of reporter gene activity was markedly decreased.



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  		Figure 4. Activity of ET-1 promoter deletion constructs in a normal diploid and heterozygous fibroblast cell lines. Equal amounts (40 µg) of the indicated ET-1 reporter constructs were transfected into TGR-1 and HET15 cells. CAT activities are displayed relative to the activity elicited by plasmid pET{Delta}875 in TGR-1 cells. Each column with a bar shows the mean ± SEM (n = 3). *, P < 0.01, pET{Delta}875 vs. deletion constructs.

 
The same constructs were also tested for trans-activation with pSPT-Myc in HET15 cells (Fig. 3Go). Truncation at -642 bp (pET1{Delta}642) eliminated trans-activation by c-Myc, except at very high c-Myc levels (80 µg/ml). Truncation at -372 bp (pET1{Delta}372) restored trans-activation at low levels of c-Myc (20–40 µg/ml), but the repression seen with pET1{Delta}875 at higher levels of c-Myc (60–80 µg/ml) was no longer observed. Deletions to -179 bp and -20 bp (pET1{Delta}179 and pET1{Delta}20) eliminated responsiveness to c-Myc, resulting in only low basal expression levels. These results indicate that two regions (-875 to -642 bp) and (-372 to -179 bp) are important for activation by c-Myc, whereas the region -642 to -372 bp is essential for repression by c-Myc.

Effect of c-Myc on ET-1 gene expression in endothelial cells
Rat fibroblast cell lines (TGR-1 and its derivatives) express the ETA receptor, but not ET-1. Therefore, the effect of c-Myc on the expression of the ET-1 gene was tested using rat endothelial cells that synthesize and constitutively secrete ET-1 (Fig. 5Go). Treatment of endothelial cells with a low dose (0.1 µM) of liposome-encapsulated c-myc antisense oligonucleotide resulted in a significant (P < 0.01) increase (132.1 ± 4.7%) in basal ET-1 release during a 24-h period, whereas higher doses (1–10 µM) led to a marked and significant (P < 0.05) inhibition of ET-1 release: 1 µM (33.5 ± 13.5%), 2.5 µM (39.4 ± 9.5%), and 10 µM (40.1 ± 19.8%), respectively. Treatment with reverse (Fig. 5AGo), random, or sense oligonucleotides (data not shown) was without effect at any concentration tested (0.1–10 µM).



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  		Figure 5. Biphasic regulation of ET-1 gene expression by c-Myc in rat aortic endothelial cells. A, Effect of c-myc antisense oligonucleotides on ET-1 secretion. Cultured rat endothelial cells were incubated with the indicated concentrations of oligonucleotides (encapsulated with Lipofectin) in serum-free medium for 24 h (see Materials and Methods). ET-1 released into medium was measured by RIA. Each column with a bar shows the mean ± SEM (n = 4). *, P < 0.05, **, P < 0.01 (reverse oligomer- vs. antisense-treated cells). B, Effect of c-myc antisense oligonucleotides on ET-1 mRNA expression. Rat endothelial cells were treated with the indicated concentrations of c-myc antisense and reverse oligonucleotides, as indicated above. Total cellular RNA (15 µg) was analyzed by Northern hybridization with ET-1, GAPDH, and ß-actin probes. All values were normalized to the internal controls (GAPDH and ß-actin). The data are plotted as relative mRNA levels of antisense-treated cells to those of reverse oligomer-treated cells. Each column with a bar shows the mean ± SEM (n = 4). **, P < 0.01, reverse oligomer- vs. antisense-treated cells.

 
Analysis of ET-1 gene expression by Northern hybridization showed that the mRNA levels in quiescent rat endothelial cells were significantly (P < 0.05) increased by treatment with lower doses of c-myc antisense oligonucleotide [0.1 µM (173.3 ± 19.6%) and 0.25 µM (144.1 ± 18.0%)], but were reduced (76.1 ± 4.5%) by a higher dose (10 µM; Fig. 5BGo). Treatment with 0.1 µM of c-myc antisense oligonucleotide also enhanced the expression of ET-1 mRNA when quiescent rat endothelial cells were stimulated with serum (data not shown). The apparent effects are underestimates of the actual expression levels, because only a fraction of the total population of cells (~50%) had taken up the oligonucleotides. We, therefore, conclude that c-Myc affects the expression of the ET-1 gene in a biphasic fashion in endothelial cells as well as in fibroblasts.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To determine whether the c-Myc protein affects the expression of the ET-1 gene, the effects of c-Myc on ET-1 gene expression were investigated in three experimental settings: 1) a panel of isogenic fibroblastic cell lines that have been genetically manipulated to stably express various levels of c-Myc protein, 2) cotransfections of reporter constructs and c-Myc expression vectors into fibroblasts with low endogenous c-Myc levels, and 3) antisense modulation of endogenous c-Myc expression in nonimmortalized endothelial cell strains. Complementary results were obtained in all three cases. We found that the ET-1 gene is regulated in a biphasic fashion: c-Myc activates at low levels and represses at higher levels.

It is well known that growth factors, cytokines, and hormones induce the expression of the immediate early class of growth-regulated genes, such as c-fos and c-myc. ET-1, a potent vasoconstrictor and mitogen, has been shown to stimulate the expression of the c-myc gene in fibroblasts and vascular smooth muscle cells (10, 37). In this communication, we demonstrate the existence of a signal transduction pathway in the opposite direction: regulation of the ET-1 gene by the immediate early transcription factor, c-Myc.

The c-Myc protein is known to activate transcription through the E-box consensus motif, CA[C/T]GTG (19), and repress transcription through the initiator (Inr) element centered around the start of transcription (27, 28). The repression is unlikely to be mediated by direct binding of c-Myc to DNA and, instead, may involve binding of c-Myc to the initiation factor TFII-I (38) and/or other proteins. Our deletion analysis of the ET-1 gene promoter revealed three distinct regions that respond to c-Myc: region A (-875 to -642 bp) and region C (-372 to -179 bp), which mediate stimulatory effects, and region B (-642 to -372 bp), which mediates a repressive effect. DNA sequence analysis revealed the presence of an E-box site (CATGTG) within region A (-656 to -660 bp). As E-box consensus sequences were not found in regions B or C, the effects of c-Myc on the expression of the ET-1 gene mediated by these regions are likely to involve other transcription factors. Although the responsiveness of ET-1 promoter to c-Myc has not been previously examined, others have also noted the presence of an inhibitory region corresponding in location to region B defined in this report (39). In agreement with their results, we have confirmed that cotransfection of pET{Delta}875 with high levels of c-Myc into rat endothelial cells resulted in repression of reporter gene activity (data not shown).

The expression of the c-myc gene is known to depend on continuous growth factor stimulation, and c-Myc mRNA and protein levels are not influenced solely by any one mitogen, but, rather, represent a composite of external proliferative influences (40, 41). These observations have led to suggestions that c-Myc may integrate several signaling pathways (30, 42). The results presented here add a further dimension to these mechanisms; the finely graded c-Myc expression levels resulting from a variety of extracellular signals would be translated into variable rates of ET-1 secretion by the cell. In this context it is of particular interest that subtle changes in c-myc expression may be sufficient to cause significant changes in ET-1 gene expression. As secreted ET-1 can act in an autocrine and/or paracrine fashion, this mechanism would be operative in normal cells, where c-Myc expression levels are in the range that activate ET-1 expression.

Our finding that c-Myc regulates ET-1 gene transcription such that it acts as an activator at a low concentration and as a repressor at a high concentration is similar to atrial natriuretic peptide (ANP) gene regulation by immediate early genes (c-fos and c-jun). Namely, experiments involving transient transfection analysis in neonatal rat cardiocytes revealed a paradoxical control of human ANP gene by c-fos and c-jun (43): c-jun increased ANP gene expression, and high levels of c-fos inhibited ANP transcription, whereas low levels of c-fos, in concert with c-jun, activated the ANP gene.

An intriguing and central question then arises as to whether there exist any pathological conditions in vivo that mirror these in vitro findings. Accumulating lines of evidence suggest that a number of factors, such as vasoconstrictive hormones (angiotensin II and vasopressin), thrombin, cytokines (interleukin-1ß, tumor necrosis factor-{alpha}, and transforming growth factor-ß) have been shown to up-regulate ET-1 gene as well as c-myc (44). These factors are also known to be responsible for the development of atherosclerosis, vascular remodeling, and cardiac hypertrophy. ET-1 conversely increases cardiac ANP gene (45) and endothelial nitric oxide production (46), both of which antagonize ET-1 action as well as inhibit ET-1 gene expression (33, 44). These counterregulatory mechanisms should prevent overshooting of ET-1 via long and short feedback loops, respectively. Thus, the autocrine feedback loop between c-Myc and ET-1 may constitute a fine counterregulatory mechanism for the maintenance of normal vascular biology, whose dysregulation could contribute to the development of vascular pathology, such as in atherosclerosis and vascular remodeling. In fact, recent reports have demonstrated the overexpression of ET-1 in atherosclerotic plaques (47) and an involvement of c-myc deregulation in abnormal proliferation and apoptosis of vascular smooth muscle cells (48).

We, therefore, propose the existence of a finely balanced autocrine/paracrine mechanism for the synthesis and secretion of ET-1, with c-Myc acting as a key intracellular effector, as illustrated in Fig. 6Go. At low levels of extracellular ET-1, basal levels of c-Myc act to stimulate the ET-1 gene, thereby increasing ET-1 release. As the rising ET-1 levels increase c-myc expression, the elevated levels of c-Myc protein act to repress the ET-1 gene, thus disrupting the autocrine feedback loop between ET-1 and c-Myc. We show that in the c-Myc expression range found in c-myc heterozygous and diploid fibroblast cells, c-Myc is an activator of ET-1 expression, whereas at elevated levels (as low as 4-fold above the diploid condition), c-Myc acts as a repressor. This may implicate a possible cross-talk mechanism between the various growth factors known to influence c-Myc expression. First, any such effector capable of stimulating c-Myc expression would initiate a feedforward loop. Second, the secreted ET-1 would act as a mitogen and further increase growth. The fact that this autocrine loop is disrupted at unphysiologically high c-Myc expression levels can be viewed as a built-in safety mechanism, such that cells with a high proliferative potential due to the overexpression of c-Myc would be rendered increasingly susceptible to apoptosis.



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  		Figure 6. A proposed model for autocrine feedback mechanism between Myc and ET-1. Boxes indicate genes; circles indicate the encoded gene products. A dashed line indicates a transfer of gene products. A straight line indicates a regulatory effect mediated by the gene product. An arrowhead indicates a positive effect; a bar indicates a negative effect. A bold solid arrow indicates a promoter.

 


   Footnotes
 
1 This work was supported in part by Grants-in-Aid from the Ministry of Education, Science, and Culture, Japan (to M.S. and Y.H.); the Ministry of Health and Welfare, Japan (to Y.H.); the Tokyo Hypertension Conference (to M.S.); the Foundation for Growth of Science (to M.S.); the Uehara Memorial Foundation (to M.S.); the Chiiki-Igaku Research Fund (to M.S.); the Tanabe Medical Frontier Conference (to M.S.); an NIH grant (to J.M.S.), and a Presidential Young Investigator Award from the NSF (to J.M.S.). Back

Received May 19, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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