©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Differentiation of Embryonal Carcinoma Cells to a Neural or Cardiomyocyte Lineage Is Associated with Selective Expression of Endothelin Receptors (*)

Juan Carlos Monge (1)(§)(¶), Duncan J. Stewart (1)(§) (2), Peter Cernacek (1)

From the (1)McGill Vascular Biology Group, Divisions of Cardiology and Medical Biochemistry, Department of Medicine, Royal Victoria Hospital and McGill University, Montreal, Quebec H3A 1A1 Canada and (2)Division of Cardiology, St. Michael's Hospital, University of Toronto, Toronto, Ontario M5B 1W8 Canada

ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Endothelins (ETs) were initially characterized as potent vasoactive peptides acting through at least two distinct receptors, ET and ET. Subsequently, their significant growth- and hypertrophy-promoting properties in cardiac and other cells were recognized. We investigated the expression of endothelin receptors during differentiation of a pluripotential embryonal carcinoma cell line (P19) to a cardiomyocyte or a neural lineage. These cells resemble those of the inner cell mass of the blastocyst, and their differentiation is believed to closely mimic critical events in early embryogenesis. Differentiation of P19 to a cardiomyocyte lineage, by aggregation and exposure to dimethyl sulfoxide resulted in induction of ET receptors as demonstrated by radioligand binding studies, Northern blotting, and reporter gene analysis. Moreover, the P19 differentiated to a cardiac lineage responded to ET-1 with a 3-fold increase in the secretion of atrial natriuretic peptide. In contrast, differentiation to a neural lineage, by aggregation and exposure to retinoic acid, was associated with the induction of predominantly ET. Therefore, selective differentiation of the P19 led to the differential expression of endothelin receptors in a pattern consistent with that observed in normal myocardial and neural tissue. The induction of endothelin receptors in a model system of early embryogenesis provides strong support for the critical role of this peptide/receptor family in differentiation and development. As well, this model system is well suited for the study of the mechanisms controlling endothelin receptor expression during differentiation.


INTRODUCTION

Endothelins (ETs)()are a family of at least three peptides (ET-1, -2, -3) with a variety of biological activities(1, 2) . ET-1 is an extremely potent constrictor of arterial and venous segments, and it also has a positive inotropic effect in isolated myocardial preparations(3, 4, 5) . In addition to its cardiovascular actions, it contracts nonvascular smooth muscle cells (bronchial, uterine, and gastrointestinal tract), it is a potent and direct stimulator of synthesis of aldosterone and atrial natriuretic peptide (ANP), and it modulates neural transmission(6, 7, 8) . Moreover, ET-1 promotes the growth of several cell types (myocardiocytes, vascular smooth muscle, fibroblasts, mesangial, and neural cells), suggesting a role in the regulation of cellular growth and tissue differentiation, as well as in pathological proliferative processes such as atherosclerosis and tumor growth(9) .

Both neonatal and adult cardiocytes show a hypertrophic response to ET-1(10, 11, 12) , as evidenced by increased protein synthesis and assembly of contractile proteins into sarcomeric units. As well, ET-1 directly stimulates the ANP gene and increases secretion of the mature ANP hormone in vitro and in vivo(13) . A number of other cardiac-specific genes have been shown to be activated by ET-1, namely those encoding myosin heavy and light chains, -actin, and troponin-T. Therefore, ET-1 has been proposed as an important paracrine mediator in the regulation of myocardial growth and development(11, 12) .

The physiological role of ETs in the central nervous system remains unclear. The presence of ET-1 and ET-3 immunoreactivity or mRNA has been established in multiple sites including the cerebral cortex, the brainstem, the hypothalamus, the basal ganglia, the cerebellum, and the spinal cord(14, 15, 16, 17, 18) . Such widespread distribution strongly suggests the possibility that ETs act as neurotransmitters, neuromodulators, or neurohormones.

The effects of ETs are mediated by specific high affinity cell surface receptors (ETR), which are distributed not only in the vasculature but also in the brain, kidney, heart, lung, adrenal cortex, uterus, and other organs. At least two types of receptors (ET and ET) have been characterized and cloned thus far(19, 20) . They differ in their distribution and in their selectivity for ET isoforms. Both have seven putative transmembrane domains similar to other members of the G-protein-coupled receptor family. The ET-1 (and ET-2) selective ET type is the predominant receptor of cardiac myocytes and smooth muscle cells. The ET receptor exhibits equal affinity for the three isoforms and is the predominant, if not exclusive, receptor in neural tissue. Both types are found in various proportions in lung and renal medulla(21) .

In the present study, we have characterized a model system of selective ET or ET receptor induction during differentiation of P19, a pluripotential embryonal carcinoma cell line (22-24), along a cardiac myocyte or a neural lineage, respectively. In view of the fact that ETs have growth- and hypertrophy-promoting activity, the demonstration of endothelin receptors in cells that resemble those of the early embryo would support a role of endothelins and their receptors in growth and differentiation.


MATERIAL AND METHODS

Cell Culture and Differentiation

P19 cells were obtained from the American Type Culture Collection (ATCC, No. CRL 1825) or kindly donated by Dr. Michael McBurney, Ottawa, Canada. Rat A10 thoracic aorta smooth muscle cells were obtained from the ATCC (No. CRL 1476). Alpha minimum essential medium and fetal bovine and newborn calf serum were from Life Technologies, Inc. All-trans-retinoic acid was from Eastman Kodak Co., dimethyl sulfoxide (MeSO) was from Sigma. Differentiation of the P19 cells to a myocyte lineage or a neural lineage was carried out by aggregation and exposure to 1% MeSO (v/v) or 0.3 µM retinoic acid, respectively, following a previously described procedure(25) .

Radioligand Binding Experiments

Intact Cells

The medium was removed from the cells growing in 24-well plates, replaced by 1 ml of sterile fresh Ham's F-12 medium containing 0.2% bovine serum albumin and 0.3% bacitracin, and the cells were allowed to equilibrate for 1 h at 37 °C. The binding of I-ET-1 or I-ET-3 (both at specific activity of 2000 Ci/mmol, Amersham Corp.) was then performed in fresh medium (0.2 ml/well) at 37 °C. In competition binding experiments, cells were incubated with 50 pM radioligand and various concentrations of unlabeled competitors (ET-1 and ET-3 from Peninsula Laboratories, Belmont, CA, BQ123 from Peptides International, Louisville, KY). The reaction was stopped by aspiration of the media. The cells were then washed sequentially with 1 ml of ice-cold 0.1% bovine serum albumin in phosphate-buffered saline (PBS) and 1 ml of PBS. After digestion in 2 N NaOH, radioactivity and protein concentration of the digest were measured by an automated gamma-counter and the Bradford assay (Bio-Rad), respectively. Nonspecific binding defined as cell-associated radioactivity in the presence of 0.2 µM unlabeled ET-1 did not exceed 10% of total radioactivity. Degradation of radioligand was monitored routinely by precipitation of the medium with 12% trichloroacetic acid. It did not exceed 4%. Similar results were found in representative experiments with high performance liquid chromatography analysis of cell-exposed media.

Membranes

Membranes were prepared from cells grown on 15-cm cell culture dishes. The cells were washed with ice-cold PBS, scraped with a rubber policeman, suspended in 0.3 M sucrose with 5 mM HEPES (pH 7.4), and homogenized on ice with a Polytron (Brinkman). The homogenate was centrifuged at 1,000 g for 10 min at 4 °C, and the supernatant was collected and centrifuged at 35,000 g for 30 min at 4 °C. The pellet was resuspended in buffer A (in mM: 124 NaCl, 5 KCl, 1 CaCl, 0.4 MgSO, 1 NaHPO, 50 HEPES, pH 7.4), aliquoted, and stored at -80 °C for usually not more than 2 weeks. In preliminary experiments, no change in binding parameters was observed under these conditions for at least 2 months.

Binding experiments were performed at room temperature for 4 h in a total volume of 0.2 ml of buffer A containing 0.2% bovine serum albumin and 0.3% bacitracin with 15-20 µg of membrane protein from unaggregated and uninduced cells, 4-8 µg from MeSO-induced cells, and 3-6 µg from RA-induced cells. The reaction was stopped by addition of 1 ml of ice-cold PBS and rapid centrifugation at 12,000 g for 3 min. In competition experiments, concentrations of I-ET-1 and of unlabeled competitors were as described with intact cells. In saturation experiments, membranes were incubated with increasing concentrations (18 to 2,000 pM) of radioiodinated ET-1 or ET-3. Nonspecific binding of I-ET-1 was assessed as above, while that of I-ET-3 was determined in the presence of 1 µM of unlabeled ET-3. Kinetic parameters (B, K) were obtained by an iterative nonlinear least-square regression analysis of bound and free radioactivity data.

Determination of Secreted Peptides

Measurement of ANP

Measurement of ANP in the media was done by a specific radioimmunoassay of SepPak-extracted media as described previously(26) . Detection limit of the assay was 0.4 pg/tube, and the intra- and interassay coefficient of variation was 7 and 11%, respectively.

Immunoreactive ET-1

Immunoreactive ET-1 was determined with the radioimmunoassay established in our laboratory (27) after extraction on SepPak-C18 cartridges (Waters Millipore, Missisauga, Ontario). After loading the media on the cartridges and washing with 10 ml of HO, peptides were eluted with 100% methanol with 75% recovery. The detection limit was 0.1 pg/tube, and the intra- and interassay coefficient of variation was 9 and 12%, respectively. Cross-reactivity of ET-3 and big ET-1 with the rabbit anti-ET-1 serum (Peninsula Laboratories, RAS 6901) was 5 and 10%, respectively.

Cloning of the cDNAs for Mouse Endothelin Receptors

Partial length cDNA fragments for the ET and ET receptors were obtained by reverse transcription-polymerase chain reaction (PCR) from total RNA from P19 cells differentiated with MeSO or RA, respectively. Oligonucleotide primers were designed, based on the rat ETR sequences (20, 28), which are complementary to highly conserved regions in the first and third transmembrane domains. The sequence of the primers was as follows: 1) ET sense, 5`-TTTTCATCGTGGGAATGGTGGG-3`; ET antisense, 5`-GACTTCTGCAAAAAGGGGAACA-3`; and 2) ET sense, 5`-GTATCATGCCTCGTGTTCGTG-3`; ET antisense, 5`CGACTCCAAGAAGCAACAGCT-3`. Amplification of human placental cDNA, bovine aorta and pulmonary artery, and rat brain and kidney cDNA yielded fragments of the predicted size (234 bp for ET and 300 bp for ET). Subsequently, the primers were utilized in reverse transcription-PCR of the P19 RNA. The conditions for reverse transcription are as published elsewhere(29) . Amplification was carried out for 40 cycles as follows: denaturing at 94 °C for 1 min, annealing at 55 °C for 1 min, extension at 72 °C for 1 min with increments of 2 s/cycle, followed by a 10-min extension after the final cycle. After chloroform extraction, the reaction was analyzed in a 2.5% agarose gel revealing products of the same size as those of the other species. The PCR products were then ligated into the PCR II vector (Invitrogen; San Diego, CA) and sequenced by the dideoxy method. Inserts were identified as endothelin receptors by sequence analysis of the GenBank data base using the BLAST program(30) . The nucleotide sequences in the amplified region were highly homologous to the respective human receptors. For subsequent experiments, fragments were excised by EcoRI digestion, purified by preparative electrophoresis and the QIAEX method (QIAGEN, Chatsworth, CA), and radiolabeled with [-P]dCTP (Amersham Corp.) utilizing T7 DNA polymerase and the random primer method with a kit from Pharmacia Biotech Inc. Specific activities of 1-3 10 dpm/µg of DNA were achieved.

Analysis of Endothelin Receptor mRNA Levels in Differentiated and Undifferentiated P19 Cells

Total RNA was isolated from the P19 cells by extraction with guanidinium thiocyanate/acid phenol utilizing the commercially available preparation RNAzol B (Tel-Test, Friendswood, TX)(31) . Poly(A) RNA was isolated by the Poly(A) tract system (Promega) and fractionated by electrophoresis on a denaturing formaldehyde agarose gel. Capillary transfer to Hybond N membranes (Amersham Corp.) was performed subsequently. RNA was cross-linked to the membranes with an ultraviolet cross-linker (Fisher Scientific, model UVXL) utilizing the preprogrammed optimal cross-link setting. For hybridization and posthybridization washes, the membranes were processed as described previously(32) . ETR probes were added in concentrations of 10 dpm/ml. Autoradiograms of the membranes were obtained with exposures of 1-3 days utilizing double intensifying screens and Kodak XAR film.

Isolation of Promoter Fragments of ETR Genes and Preparation of Luciferase Expression Plasmids

Fragments containing approximately 850 bp of the 5`-flanking region and 90 bp of the first exon of the human ET gene (33) and 1.1 kilobase pairs of the flanking region and 250 bp of the first exon of human ET gene (34) were obtained by PCR of human genomic DNA as described previously(35) . The primers employed were for ET, 5`-GTCGGTACCGGATCCTCCAGCCCCTGCTACAT-3` (sense) and 5`-GCTCTCGAGTGTCCTCCCCGTCTCCTCCCAAA-3` (antisense); and for ET, 5`-GCTGGTACCGTGCGTGATAACTTGCCCTTG-3` (sense) and 5`-GCTCTCGAGTAGTGGGTGGCGTCATTATCT-3` (antisense). A KpnI site was included in both sense primers, whereas XhoI sites were incorporated into both antisense primers. After digestion with the appropriate enzymes to generate the restriction sites, the fragments were inserted into the pGL2 enhancer vector (Promega). Sequences were confirmed by dideoxy sequencing in the double-stranded plasmids.()Large scale preparations of expression plasmids for transfection were performed by the method of QIAGEN Inc.

Transfection of P19 Cells

Expression plasmids were transfected into differentiated and undifferentiated P19 cells by employing cationic liposomes and serum-free conditions (Lipofectin and Opti-MEM respectively; Life Technologies, Inc.) and following the protocols of the manufacturer. Transfection conditions were optimized with the reporter plasmids pSV--galactosidase and pGL2-control (Promega) in which the expression of the -galactosidase and firefly luciferase genes, respectively, are regulated by the SV40 early promoter and enhancer. Optimal transfection conditions for a 35-mm dish were 10 µg of Lipofectin, 1-5 µg of plasmid DNA, and 6 h of incubation in serum-free conditions. The peak levels of transient expression of the reporter genes was observed at 48 h (not shown). These conditions were utilized in all subsequent experiments with the ETR promoter constructs. -Galactosidase activity was assayed by a colorimetric assay(36) . Luciferase was quantified in a Berthold AG luminometer according to a previously described protocol(37) .


RESULTS

Binding of Radioiodinated Endothelins

The intact cells growing in monolayers and not subject to aggregation exhibited a barely detectable binding of 50 pMI-ET-1. After aggregation, and more markedly after induction with RA or with MeSO, there was a clear increase of binding. In the competition experiments (Fig. 1), unlabeled ET-1 readily displaced I-ET-1 in both RA- and MeSO-induced cells with similarly high affinity (IC of 80 and 110 pM, respectively). While the linear Scatchard plots derived from these data suggested an apparent homogeneity of the binding sites, the affinity profile of BQ123 revealed its heterogeneous nature. This ET-selective antagonist was a very weak competitor in RA-induced cells (IC almost 100 µM), while it potently displaced the radioligand in MeSO-induced cells with IC 1000 times lower. Hence in MeSO-induced cells, the majority of ET-1 binding was due to ET receptors, while the RA-induced cells expressed mostly ET receptor. The similarity of the affinity profiles of BQ123 and ET-3 obtained in rat aortic smooth muscle cells (not shown), known to express almost exclusively the ET receptor type with the profile found in MeSO-induced cells, supported this notion.


Figure 1: Competition with binding of I-ET-1 by cold ET-1 (circles), ET-3 (squares), and BQ123 (triangles) in MeSO-induced (full line) and RA-induced (dashed line) P19 cells.



Membranes were then obtained from five different preparations of each of the following, nonaggregated, aggregated, MeSO-induced, and RA-induced cells, and were used to extend the competition binding studies, as well as to examine the density of binding sites (B) both with I-ET1 and I-ET3. The increase in B of ET-1 binding as assessed by Scatchard analysis of competition binding data (Fig. 2) was 20-, 100-, and 180-fold in aggregated, MeSO-induced, and RA-induced cells, respectively, with K values in the range of 40 to 150 pM. When the time course of binding of I-ET-1 and I-ET-3 was compared (Fig. 3), RA-induced cells bound both radioligands almost equally, while MeSO-induced cells bound only I-ET-1. Affinity profiling with ET-1, ET-3, and BQ123 gave similar results as those obtained in the intact cells, thus confirming the prevalence of ET-type receptors in MeSO-treated cells and that of ET type in RA-induced cells. Saturation binding experiments both with I-ET-1 and I-ET-3 also showed that the majority of ET-1-binding sites (60-90% in various preparations) in RA-induced cells belong to ET type. In contrast, MeSO-derived membranes bound almost exclusively radiolabeled ET-1, again compatible with a strikingly prevailing presence of ET type receptors (Fig. 4).


Figure 2: Scatchard plot of competition binding data obtained with I-ET-1 as radioligand and cold ET-1 as competitor in membranes from control (nonaggregated, diamonds, and inset), aggregated (triangles), MeSO-induced (squares), and RA-induced (circles) P19 cells.




Figure 3: Time course of binding of I-ET-1 (thick line) and I-ET-3 (thin line) in membranes obtained from MeSO-induced (squares), and RA-induced (circles) cells. The concentration of both ligands was 50 pM. The experiment was repeated three times; a representative result is presented.




Figure 4: Binding of increasing concentrations of I-ET-1 (circles, squares) and -I-ET-3 (triangles), MeSO-induced (squares), and RA-induced (circles, triangles) P19 cells. Binding of I-ET-3 in MeSO-induced cells was barely detectable.



Secretion of ET-1 and ANP by P19 Cells

To ascertain whether the apparent density of binding sites could be affected by prior receptor occupancy by ET-1 secreted by the cells, immunoreactive ET-1 levels were measured in the conditioned media (Fig. 5A). After a 24-h incubation, the highest secretion levels were found in undifferentiated cells, but never exceeded 5 pM, corresponding to secretion rate of 90-100 pg/mg of cell protein/24 h. At this concentration, ET-1 inhibited binding of radioligand by <5% in competition studies (Fig. 1). Hence, the effect of the endogenous peptide on the results of binding experiments was negligible even in the cells with the highest secretory activity, i.e. undifferentiated P19. Aggregation alone or associated with MeSO induction markedly decreased ET-1 secretion. The cells induced with RA secreted only 10% of the amount produced by undifferentiated cells. To evaluate the functionality of the receptor induced during differentiation, we also measured secretion of ANP and its response to ET-1 added to the media (Fig. 5B). Negligible secretion of ANP in undifferentiated cells was not affected by addition of ET-1. Aggregation alone resulted in a slight increase in ANP production, but again without any response to ET-1. In contrast, the highest secretory rates were observed in the cells differentiated to a myocyte lineage and, more importantly, only these cells exhibited a marked (3-fold, p < 0.01) increase in ANP secretion in the presence of ET-1. This response, characteristic for cardiac myocytes in vivo and in vitro, suggests that ET receptors induced in these cells were functional and coupled to the appropriate signal transduction mechanisms.


Figure 5: A, secretion of ET-1 in P19 cells. Mean ± S.E. of triplicate measurements is presented. *, significantly different from control (nonaggregated) P19 cells. B, secretion of ANP in P19 cells in the absence and presence of ET-1 (10M). Mean ± S.E. of triplicate measurement is presented. *, significantly higher than in the absence of ET-1.



Expression of ETR mRNA during Differentiation

The levels of ETR mRNA determined by Northern blotting were insignificant in the exponentially growing cells and in cells subjected to the aggregation protocol without inducing agents. In contrast, selective increases in ETR mRNA were detected in the differentiated cells, specifically a marked predominance of ET mRNA after RA induction and exclusively ET after induction with MeSO (Fig. 6). No difference was observed in the mRNA levels of the constitutively expressed glyceraldehyde-3-phosphate dehydrogenase between undifferentiated and differentiated P19 cells. These findings are in complete agreement with those of the receptor binding studies and support our conclusion that competition by secreted ET-1 in the media is not the explanation for the negligible binding of ET-1 to the undifferentiated cells.


Figure 6: Northern blotting of mRNA from P19 cells (10 µg/lane): 1, monolayers of exponentially-growing undifferentiated P19 cells; 2, P19 aggregated in the absence of inducers; 3, P19 differentiated to a neural lineage; 4, P19 differentiated to a cardiomyocyte lineage.



Luciferase Expression in Differentiated P19 Cells Directed by ETR Promoters

To evaluate further the mechanisms responsible for the expression of the ETR genes during differentiation of the P19 cells to a myocyte or a neural lineage, we constructed expression plasmids in which the expression of the firefly luciferase gene was driven by fragments from the human ET or ET promoters. The major putative binding sites for transcription factors present in the ET promoter fragment are: a CArG box, an inverted MyoD-E2A binding site, an SP-1 binding site, and four GATA motifs. The ET promoter fragment contains the following putative binding sites, a GATA motif, four ``E boxes,'' an acute phase reactant regulatory element (APRRE), and an inverted APRRE. The human ET promoter was able to direct the expression of luciferase in the MeSO-induced cells and to a much lesser degree in the RA-induced cells. In contrast, the human ET construct directed the expression of luciferase only in the RA-induced cells (Fig. 7). Minimal luciferase activity was observed with all constructs in the undifferentiated cells. These results were in complete agreement with the mRNA and binding data and are interpreted as indicating that, in least in part, the induction of expression of the ETR genes during differentiation of the P19 cells is transcriptionally regulated. Mock transfected P19 cells of all types, as well as cells transfected with promoterless luciferase plasmids, expressed levels of luciferase which were, in most instances, not significantly different from the background of the luminometer.


Figure 7: Luciferase activity (RLU) in differentiated P19 cells (clear bars, cardiomyocyte lineage; shaded bars, neural lineage) transfected with ETR promoter constructs (hETAR, ET promoter-luciferase; hETBR, ET promoter-luciferase). *p < 0.01 compared to the other bars of the same shading. , MeSO; , RA.




DISCUSSION

Endothelins (ET-1, -2, and -3) were initially characterized as a peptide family with marked effects on vascular tone(1, 2) . Besides their potential role in cardiovascular regulation, these peptides were also shown to elicit mitogenesis and stimulate the synthesis of DNA in a broad range of cardiovascular and noncardiovascular cells, such as vascular endothelial and smooth muscle cells, cardiac myocytes, fibroblasts, glomerular mesangium, and neural cells(38) .

The physiological role of endothelins remains unclear. Expression of ET-1, for instance, is very low in the plasma and tissues of normal adult organisms but is induced to a very significant extent during extreme pathophysiological conditions such as acute myocardial infarction, cardiogenic and septic shock, pulmonary hypertension, and idiopathic pulmonary fibrosis(39, 40, 41) . In contrast, significant expression of ET-1 has been reported in normal embryonic and fetal tissues raising the possibility that the endothelin family may play a role in differentiation, growth, and development(42) .

While ET appears to be the only receptor expressed in neural tissue, both ET and ET have been observed in the heart(21) . In situ hybridization analysis of receptor mRNA revealed the expression of ET- and ET-specific transcripts (with a predominance of the former) both in the atria and ventricles(21) . Binding experiments with a selective ET antagonist (BQ123) showed, as well, that the majority of binding sites on cardiac myocytes belong to the ET class(43) .

To test the hypothesis that cells at various stages of development or differentiation would express ET receptors differentially, we utilized the mouse embryonal carcinoma cell line P19. In view of their antigenic and biochemical resemblance to the cells of the inner cell mass of the blastocyst(44) , these cells have been widely used to study the events occurring during the early development of the mammalian embryo. To the best of our knowledge this is the first report on the predominant, and nearly selective, expression of ET and ET receptors during differentiation of a pluripotential cell line toward a cardiac myocyte or a neural lineage, respectively.

In the classical receptor binding experiments we demonstrated that the process of differentiation was associated with a striking increase in the density of binding sites for ETs in both the myocyte and the neural lineage. Competition with the specific ET antagonist BQ123 established that the vast majority of the receptors expressed during differentiation of the cells in the presence of MeSO (myocyte lineage) are of the ET type. In contrast, the binding of labeled ET-3 and competition with unlabeled ET-3 determined that there was a marked predominance of ET receptors in the cells induced to differentiate to a neural lineage with retinoic acid. In these cells a second type of receptor, presumably ET, was also present but accounted for no more than 10-40% of the binding sites.

Thus, upon differentiation into the two different lineages, the P19 cells expressed the predominant receptors observed in myocardium (ET) or in neural tissue (ET)(21) . Moreover, differentiation of P19 cells toward a myocyte lineage resulted in the induction of ANP secretion, which was increased in response to ET-1, an ET-mediated phenomenon well known to occur in normal cardiomyocytes(12, 45) . ET-1-responsive secretion of ANP does not by itself establish the cardiac nature of the MeSO-induced cells as ANP is produced in other tissues. However, their response to ET-1 suggests that the ET induced are functional and coupled to appropriate signal transduction mechanisms.

Further evidence supporting the selective pattern of receptor induction was provided by Northern blotting analysis of P-19 mRNA. ET mRNA was observed only in the RA-induced cells and was indeed the most abundant of the ETR mRNAs observed in these studies. Visualization of ET mRNA, in contrast, required longer exposure times in both the RA- and MeSO-induced cells. In the latter only ET mRNA was detectable.

Our reporter gene experiments with firefly luciferase suggest that the differentiation of the P19 cells is associated with selective transcriptional activation of the human ETR genes. Thus, the differentiation of the P19 cells constitutes an excellent model system to study the expression of ETR during differentiation, in particular, and the regulation of ETR expression in general. Ongoing studies utilizing this model system may allow us to identify the cis- and trans-acting factors that control the selective induction of these genes.

Furthermore, the striking level of induction of endothelin receptors observed during the differentiation of the P19 cells and the high affinity of the receptors (K values of 40-150 pM) strongly support a physiological role for the ETR in these embryonal cells. The importance of ETR in embryogenesis has been underscored by recent studies of targeted disruption of the ET-1 and ET genes. Mice homozygous for the disruption of either gene exhibited severe developmental abnormalities of structures derived from the first pharyngeal arch and died shortly after birth of respiratory failure(46, 47) . Moreover, following ET disruption a high incidence of abnormal cardiac development was observed, with at least 50% of animals demonstrating membranous ventricular septal defects.()

Further support for the role of endothelins and their receptors in mammalian development has been provided by three very recent reports on ET and ET-3. The human ET gene was identified as the recessive susceptibility locus for Hirschprung's disease in chromosome 13q22 (48). An absence of enteric ganglia in the distal colon and a failure of innervation in the gastrointestinal tract are characteristic of this disease. Moreover, in patients with Hirschprung's disease a G T mutation was identified in exon 4 of the ET gene resulting in the substitution of the highly conserved Trp-276 residue in the fifth transmembrane domain of the receptor with a Cys residue. This mutation produces a receptor with an impairment of ligand-induced responses. Additionally, the targeted (gene knockout) and natural (piebald-lethal) mutations of mouse ET both produce aganglionic megacolon, analogous to the human Hirschprung's disease, and abnormalities in epidermal melanocytes leading to a spotted coat color(49) . Finally, the targeted disruption of the mouse ET-3 leads to a similar, although slightly less severe, phenotype of megacolon and color spotting(50) . A natural recessive mutation that results in the same developmental defects in mice, lethal spotting, is associated with a point mutation of the ET-3 gene, which replaces the Arg residue at the C terminus of big ET-3, an inactive precursor, with a Trp residue and prevents the generation of mature ET-3(50) .

Hence, endothelins and their receptors play a critical role in specific aspects of mammalian differentiation and development. ET-1 and its specific receptor ET are important in the differentiation of tissues derived from the first branchial arch, particularly those in which cell lineages of the cephalic neural crest play an essential role. As well, ET-3/ET interactions contribute to the normal development of two additional neural crest lineages, the vagal neural crest-derived enteric neurons and the trunk neural crest-derived epidermal melanocytes. Our studies demonstrate the selective induction of ETR in a process believed to mimic, morphologically and biochemically, some of the critical events of early embryogenesis. Thus, the present report provides a model system well suited for the study of the cellular mechanisms that control the selective expression of ETR during differentiation into two highly relevant cell lineages.

In view of the importance of the endothelin system in differentiation of myocyte and neural tissues, a better understanding of the transcriptional events which control the tissue specific expression of ETR is essential for a broader comprehension both of normal developmental events and the perturbations which produce congenital abnormalities.


FOOTNOTES

*
This work was supported by a grant for a ``Equipe de Recherche Multidisciplinaire'' of the Fonds de la Recherche en Santé du Québec. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported as a Chercheur-Boursier by les Fonds de la Recherche en Santé du Québec.

To whom correspondence should be addressed: Division of Cardiology, St. Michael's Hospital, 30 Bond St., Toronto, Ontario M5B 1W8, Canada. Tel: 416-864-5970; Fax: 416-864-5336.

The abbreviations used are: ET, endothelins; ETR, ET receptor; ANP, atrial natriuretic peptide; RA, retinoic acid; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; bp, base pair(s).

The nucleotide sequence of the promoter fragments of the human endothelin receptor genes can be retrieved from the GenBank/EMBL Data Bank with accession numbers ET, D11144, and ET, D13162.

M. Yanagisawa, personal communication.


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