blasts and can affect the epithelial-mesenchymal transfor- mation of Madin-Darby canine kidney epithelial cells and generate supernumerary axial structures in ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 268,No. 19, Issue of July 5 , pp. 14404-14411, 1993 Printed in U.S.A.
Identification of an Extracellular 130-kDa Protein Involved in Early Cardiac Morphogenesis* (Received for publication, February 19, 1993)
Mehrdad RezaeeS, Keitaro Isokawat, Nadine Halligan, Roger R. Markwaldll, and EdwardL. KrugflII From the Department of Cellular Biology and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin53226
Previous studies indicate that the transformationof system and secretory granules. By polymerase chain cardiacendotheliuminto mesenchyme is dependent reaction analysis,the message for ES/130 was detected upon a developmentally regulated signal expressed by in the developing heart just prior to and duringmesits associated myocardium. This process can be mim- enchyme formation. These results are consistent with at a critical step in the initiation icked in culture by substituting a non-cytolytic EDTA ES/130 being involved extract of embryonic heart tissue for the presenceof of the epithelial-mesenchymal transformation of carmyocardium. Polyclonal antibodies (ES 1) generated diac endothelium. against the EDTA-extractable proteins both localized tothecardiacextracellularmatrixprecedingthe transformation of endothelium and blocked this process in culture. Based on these observations, we hypothThe morphological transformation of closely associated epesized that ES1 antigens participate in the formation ithelial cells into adispersed population of mesenchyme is an of cardiac mesenchyme. essential event in remodeling the primitive heart tube into a The present study was undertaken to prepare cDNA multichambered organ. Early in development, the heart conand antibody probes for individual ES1 antigens to sists of two concentric epithelial cell layers, an outer myocarbetter characterize their involvement in this important dium andinner endothelium, which areseparated by an morphogenetic event. An expression library was conacellular, expansive extracellular matrix (ECM),’ also referred structed in Uni-ZAP using poly(A+) RNA from embryto as “cardiac jelly” (1).Later in morphogenesis, a competent onic cardiocyte cultures that had been shown previously to secrete proteins that engender the formationpopulation of cardiac endothelial cells undergo an epithelialof cardiac mesenchyme. Screening of this expression mesenchymal transformation characterized by cell hypertrolibrary with ES1 antibodies resulted in several clones, phy, loss of cell-cell adhesion molecules, and increased expresone of which (“ES1-2.la”) is described in this report. sion of substrate adhesion molecules and urokinase (2-6). ES1-2.la has a 2.6-kilobase pair insert, the sequence These “activated” endothelial cells acquire a fibroblastic morphology and subsequently invade the adjacent ECM to form of which exhibits no apparent homology to those in data banks. A fragment (852 base pairs) from the 5’ cardiac cushion tissue, which contributes to valvular and septal tissues of the mature heart (7). region of ES1-2.la cDNA was subcloned intothe It has been shown previously that theepithelial-mesenchyexpression vector pGEX-ST, and a 20-kDa fragment of the resulting proteinused to prepare affinity-puri- mal transformation of cardiac endothelium in culture is dependent upon the presence of myocardium and its associated fied antibodies. Immunoblotting detected a 130-kDa protein YES/ ECM (8-10). Further studies revealed that ECM proteins in 130”)in two preparations that elicit mesenchyme for- non-cytolytic EDTA extracts of embryonic hearts and the mation, Le. EDTA extracts of embryonic hearts and conditioned medium of cultured embryonic cardiocytes could conditioned medium of cardiocyte cultures. Functional effectively substitute for co-culture with myocardium (11,12). Recently, an antiserum (ES1) was generated against the prostudies showed that antibodies to ES/130 inhibited the epithelial-mesenchymal transformationof cardiac en- teins present inthe 100,000 x g pellet fraction of heart EDTA dothelium in culture. Immunohistochemistryof cardi- extracts; this preparation elicits, and ES1 antibodies block, the formation of mesenchyme from cardiac endothelium in ocyte cultureslocalized ES/130 protein to the vacuolar culture (3, 13). ES1 antigens are associated as multicompo* This work wassupported in part by National Institutes of Health nent, particulate complexes in vitro and can be observed in Grants HL44928 and HL33756 and American Heart Association of vivo to associate with fibronectin within the myocardial baseWisconsin Grant 89-GA-78. The costs of publication of this article ment membrane (3, 13, 35). Immunohistochemical studies were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with indicate that ES1 antigens are expressed transiently during morphogenesis and are restricted to regions undergoing pu18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequencefs)reported in thispaper has been submitted tative inductive interactions (13). Based on these data we to the GenBankTM/EMBL Data Bank withaccessionnumber(s) concluded that one or more of the proteins recognized by ES1 L13973. $Supported by Fellowship 90-FA-15 from the American Heart Association of Wisconsin and the Medical College of Wisconsin Medical Scientist Training Program. Present address: Dept. of Anatomy, Nihon University School of Dentistry, 1-8-13 Kanda-Surugadai, Chiyoda-Ku, Tokyo 101, Japan. T Present address: Dept. of CellBiology, Medical University of South Carolina, 171 Ashley Ave., Charleston, SC 29425-2204. I( T o whom correspondence should be addressed. Tel.: 803-7921543: Fax: 803-792-0664.
The abbreviations used are: ECM, extracellular matrix; BCIP, 5bromo-4-chloro-3-indolyl phosphate; BSA, bovine serum albumin; EBSS, Earle’s balanced salt solution; IPTG, isopropyl-@-D-thiogalactopyranoside; NBT, nitro blue tetrazolium; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; TGF-B, transforming growth factor (3; bp, base pair(s); MOPS, 4-morpholinepropanesulfonic acid; AV, atrioventricular canal; PBS, phosphate-huffered saline; BSA, bovine serum albumin.
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ES/130 in Developing Chick Heart is a myocardially derived signal that elicits endothelial transformation into mesenchyme. In order to better understand the role of these ECM proteins in cardiac morphogenesis and the molecular basis of their interaction with each other and fibronectin, individual ES1 antigens need to be identified and characterized. HOWever, due to the heterogeneity of proteins recognized by ES1 antibodies and the paucity of starting material for conventional purification,we pursued an “epitope selection” strategy (14-16) of screening an expression library with the polyspecific ES1 antiserum to identify individual antigens. In this report, we present the partial cDNA and deduced amino acid sequence of one ES1 antigen (termed ES/130) obtained from an embryonic chicken cardiocyte expression library. This protein has a molecular mass of 130 kDa and is present intwo preparations that elicit mesenchyme formation from cardiac endothelium, i.e. the EDTA extractof embryonic heart tissue and cardiocyte conditioned medium. In addition, antibodies to ES/130 block the epithelial-mesenchymal transformation of cardiac endothelium in culture. Based on these data and computer homology searches, ES/130 appears t o be an as yet uncharacterized protein that may be involved in initiating cushion tissue formation. MATERIALS AND METHODS
Expression Library Preparation and Screening-An embryonic heart expression library was prepared in Uni-ZAP XR (Stratagene, San Diego, CA). Primarycultures of embryonic cardiocytes were established from the hearts of 2100 stage 14-16 chick embryos (17) as described previously (12). On day 7, the cell layers were rinsed briefly with EBSS, harvested by scraping, homogenized in guanidine isothiocyanate, and total RNA isolated by centrifugation through cesium trifluoroacetate (18). Poly(A+) RNA was isolated from the total RNA by oligo(dT)-cellulose chromatography (Pharmacia LKB Biotechnology Inc.). The resulting poly(A+) RNA (3-5 pg) was sent to Stratagene for custom library construction in Uni-ZAP XR. The cDNAs (average size >500 bp) were inserted in a sense orientation (5’-EcoRI/B’-XhoI) with respect to the h c Z promoter. The library was screened according to Stratagene’s recommended protocol using XL1-blue host cells, the IgG fraction of ES1 antiserum, and alkaline phosphatase-conjugated goat anti-rabbit IgG (Bio-Rad). ES1-positive plaques were visualized with BCIP/NBT (Sigma),purified, and stored in 5% chloroform. pBluescript phagemids from ES1-positive clones were rescued by in vivo excision (19) in the presence of R408 helper phage (Stratagene) and used to transfect XL1-blue host cells. Ampicillin resistance colonies contained the pBluescript double-stranded phagemid with the cloned cDNA insert. The presence and size estimation of the cDNA insert was determined by restriction analysis. PstI fragments of the recombinant plasmid ES1-2.la were subcloned into pBluescript for DNA sequencing by the dideoxy chain termination method (20) using Sequenase version 2.0 (U. S. Biochemical Corp.). Additional sequence data were obtained using synthetic oligodeoxynucleotide primers (Operon). The consensus sequence presented in Fig. 2 was derived from 171 overlapping contiguous sequences (83 coding strand and 88 complementary strand contiguous sequences). Nucleotide fragments were assembled and translatedusing the sequence analysis software package of the Genetics Computer Group (Madison, WI); homology searches were performed by FASTA analysis with the GenEMBL data base default options (21). RNA Amplification-Total RNA was purified from embryos with RNAzol (Cinna/Biotecx, Friendswood, TX) according to the manufacturer’s protocol and quantitated by the absorbance a t 260 nm. cDNA was synthesized using 1-5 pg of total RNA/reaction in 50 pl of a solution containing 0.2 mM dNTPs, 0.2 M dithiothreitol, 400 units of Moloney murine leukemia virus reverse transcriptase (Bethesda Research Laboratories), 20 units of RNasin (Promega),70 mM KCI, 3 mMMgC12, and 0.5 p M antisense oligonucleotides (22). After 1h at 37 “C,each reaction received 50 p1 of 12 mM KCI, 0.01% gelatin, 1.0 p M sense and 0.5 p M antisense oligonucleotides, and 2.5 units of Taq DNA polymerase (Perkin-Elmer Cetus). The amplification program included denaturation at 94 “C for 5 min, 30 cycles of annealing a t 55 “cfor 30 s, extension at 72 “C for 30 s, denaturation at 94 “C
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for 1.5 min, and a lo-min extension cycle at 72 “C. Reaction products were analyzed by agarose gel electrophoresis, in conjunction with susceptibility to restriction enzymes, and by dot and Southern blotting (23). Northern Blotting-Poly(A+) RNA wasisolated from stage 16 total embryos or dissected hearts, using the Micro-Fast Track kit (Invitrogen). Northern blottingwas done essentially as described by Maniatis et al. (23). Duplicate RNA samples were subjected to electrophoresis in MOPS-formaldehyde running buffer, and the separated RNAs were blotted onto nitrocellulose and then probed with 35S-labeled cRNA probes. cRNA probes were constructed by using an RNA transcription kit (Stratagene) according to the manufacturer’s protocol. Antibody Generation-ES1-2.la cDNA (in pBluescript) was digested with SmaI and HincII (Molecular Biology Resources, Milwaukee, WI). (The SmaIsite is 12 bp 5’ to the EcoRI site; therefore, 12 bp ofpBluescript and 16 bp from the linker (ligated during the library construction) sequences were included.) The 852-bp fragment (2.la’) from the 5’-end of ES1-2.la was ligated into pGEX-2T (Pharmacia; cut with SmaI), placing the insert in frame with both the hcZ promoter and glutathione S-transferase sequences upstream to the cloning site (24). The insertion site of the 2.la’ cDNA in pGEX-2T was confirmed by sequence analysis with 2.la”specific primers. The resulting pGEX-2T-2.la’ recombinant vector was used to transform competent XL1-blue host cells. Fusion protein expression was induced in mid-log phase cultures with 1 mM IPTG for 2 h at room temperature. Isolation of the glutathione S-transferase-2.la’ fusion protein was performed by glutathione-agarose affinity chromatography (24). Briefly, cells were collected by centrifugation at 10,000 X g for 5 min at 4 “C, suspended in PBS (20 mM sodium phosphate, pH 7.3, containing 150 mM NaCl), sonicated on ice for 30 s, Triton X-100 added to 1%final concentration, re-centrifuged, and thesupernatant adsorbed onto glutathione-agarose beads (sulfurlinked; Sigma). After extensive washing, the bound glutathione S transferase-2.la’ fusion protein was eluted with 10 mM reduced glutathione. The 2.la’ polypeptide was cleaved from glutathione S transferase by treatment with 100 ng of human thrombin (Sigma)/ 50 pg of fusion protein (30 min at 25 “C). Centrifugal ultrafiltration (Ultrafree-MC, 10,000 molecular weight cutoff; Millipore) was used to separate 2.la’ protein (filtrate) from glutathione S-transferase and uncleaved fusion protein. Amino acid sequence analysis was performed on about 100 pmol of the 2.la’protein, which had been transferred (25) onto Immobilon-P membrane (Millipore) using an Applied Biosystems 477A protein sequenator. After collection of preimmune serum, a New Zealand White rabbit was injected with 10 pgof purified 2.la’ protein in RIBI adjuvant (RIBI Research Inc., Hamilton, MT). The rabbit was boosted twice a t 3-week intervals with 10 pg of 2.la’ protein in RIBI adjuvant, and immune serum obtained every 2-3 weeks thereafter. Affinity-purified antibodies were prepared with glutathione S-transferase-2.la’ fusion protein covalently bound to membrane affinity chromatography discs (Millipore) according to themanufacturer’s protocol. Western Blotting-Tissue homogenates were prepared by sonication of whole or dissected regions of embryos for 20 s in ice-cold 10 mM Tris-HCI, pH 8.0, containing 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 p~ pepstatin A, and 2 p~ leupeptin. In addition, intact embryonic hearts (stages 15-16) were incubated with EDTA to extractmatrixproteins as described previously (12). Samples containing 10-15 pg of protein ( A Zrelative ~ to IgG) were solubilized in 63 mM Tris-HCI, pH 6.8, containing 2% SDS, 6 M urea, and 40 mM dithiothreitol, boiled for 3 min,subjected to SDS-polyacrylamide electrophoresis (26), and blotted onto Immobilon-P membrane (Millipore) (25). Nonspecific binding to themembranes was blocked with 20 mM Tris-HCI, pH 9.0, containing 150 mM NaCl and 5% BSA, incubated with primary antibodies, rinsed with TBSS-T-BSA (20 mM Tris-HCI, pH 7.4, containing 500 mM NaCl, 0.05% Tween 20, and 1%BSA), and incubated with alkaline phosphatase-conjugated goat anti-rabbit IgG (Bio-Rad) in TBSS-T-BSA. Positive bands were visualized with BCIP/NBT in 100 mM sodium carbonate buffer, pH 9.8, containing 10 mM MgCl2. Immunohistochemistry of Cultured Embryonic Cardiocytes-Cardiocyte cultures were prepared from embryonic stage 15-16 chick hearts (17) essentially as described previously (12). Hearts were collected in Earle’s balanced salt solution, incubated in calcium/ magnesium-free Dulbecco’s phosphate-buffered saline containing 5 mM EDTA for 30 min at 4 “C, and dissociated into single cells with 0.5% porcine pancreatic trypsin for 10 min at 37 “C. The resulting cells suspension was exchanged into medium 199 (containing 1%
ES/130in Developing Chick Heart
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chicken serum, 5 pg/mlinsulin, 5 pg/ml transferrin, 5 ng/ml selenium, andantibiotics),platedontogelatin-coated35-mmtissueculture plastic, and the medium replaced every 2 days. Cultures were processedfor immunohistochemistry by washing with EBSS, fixing in 70% ethanol for 15 min, then in ether:ethanol (1:l;v/v) for 30 min, washing with 70% ethanol, then 50% ethanol for 30 min each, distilled water for 15 min, and PBSfor 30 min. The cultures were treated with 1% BSA in PBS for 1 h, followed by incubation in primary antibody and fluorescein-conjugated goat antirabbit IgG (Cappel) for 1 h each, mounted in90% glycerol, 10% PBS containing 0.2 M n-propylgallate (27), and observed with a Nikon Optiphot epifluorescent microscope. Tissue Culture-The functional involvement of ES/130 in cardiac endothelial differentiation was tested using a culture bioassay described previously (13).Briefly, fertilized eggs from White Leghorn chickens were incubated a t 37.5 “C and 60% relative humidity to obtain stage 14-minus embryos (17). The AV regions (consisting of myocardium, associated myocardial basement membrane, and endothelium) wereremoved,placed immediatelyontothesurface of drained collagen gels (3-4 explants/well), and incubated for 2 h to allow attachment of the explants onto the collagen gel surface. At this point, the ES/130 IgG (extensively dialyzed against medium 199) was added at different concentrations (diluted in medium199) to separate cultures. After 24 and 48 h, the cultures were observed with a Leitz inverted microscope and “optically sectioned” by changing focal planes to reveal any mesenchymal cells beneath the surface of the endothelial monolayer. RESULTS
28s-
18s-
FIG.1. Northern blot analysis of ES1-2.la expression in the total embryo and embryonic heart tissue. Poly(A+) RNA fromstage 15-16 hearts (2 pg/lane, A and C) and stage 16 total embryo (2 pgllane, R and D )were separated by denaturing (formaldehyde) agarose (1%)gel electrophoresis. The separated RNAs were blotted onto nitrocellulose and probe with ”S-labeled antisense ( A and B ) or sense (C and D )riboprobes.
An expression library was constructed with poly(A’) RNA isolatedfrom primary cultures of embryonicchick hearts. These cultureswere used as the RNA sourcefor the following a potent similarity did not represent characteristic or functional patreasons. 1)Conditioned media from these cultures is inducer of theepithelial-mesenchymaltransformation,an terns for thosemolecules. However, molecules with potential effect which is blocked by ES1 antibodies (13); 2) greater biological relevance, i.e. chicken sequences,growthfactors, quantities of starting material canbe harvested from cultures gene regulating factors, muscle-specific molecules, and extramuch more rapidly than individually dissected hearts, thus cellular proteins, were examined in greater detail. These se5 X lo5 quences were compared individually with the ES1-2.la numinimizing mRNAdegradation.Afterscreening plaques, several plaques that reacted with the ES1 antibodies cleotide and amino acidsequencesfrom all possible open were obtained.The cDNAfrom one of the ES1-positive reading frames of the ES1-2.la using BESTFIT and GAP plaques, called “ES1-2.la,” is thefocus of this report. programs, respectively. In none of the cases examined did Northern blot analyses indicated the presence of an mRNA regions homologous at the nucleotide level correlate with the for ES1-2.la in heart tissue from embryos just prior to the open reading frameshown in Fig. 2. Homologies at theamino onset of mesenchyme formation (Fig. 1).Only one transcript acid level did not spanmore than 4 amino acids and exhibited was discernible, whichwas about 3.5 kilobases in size. EcoRI/ no consensus oridentifiable functional patterns. XhoI restriction digestion of ES1-2.la in pBluescriptshowed The deduced amino acid sequence of ES1-2.la was examthat the insert for this clone was about 2.7 kilobase pairs ined for patterns characteristic of post-translational modifi(data not shown). Therefore the ES1-2.la clone represents cation and structural motifs. MOTIF analysis detected several approximately 80% of the full-length mRNA for this protein. common modification sites in ESl-Z.la,i.e. 1N-glycosylation DNA sequence analysis of ES1-2.la revealed an insertsize site, 1 cAMP/cGMP-dependent protein kinase phosphorylaof 2600 bp (Fig. 2). The largest open reading frame of the tion site, 2 N-myristylation sites, 11 protein kinase C phoscDNA insert extends from the @-galactosidase promoter of phorylation sites, and 18 casein kinase I1 phosphorylation the vector for 2177 bp and would encode a polypeptide of sites. No significant structuralmotifs were found by PROFI81,002 daltons; thetwo other potential reading frames contain LESCAN analysis. However, manual inspection of the ES1multiple stop codons. The first deduced methionine in ES1- 2.la aminoacid sequence revealed a predictable periodicity of 2.la occursat position 507, but it islikely that the transcrip- lysineresidues (i.e. K(X)5-6Koccurs16 times, commonly tion start site occurs in the 5‘ mRNA sequence not accounted starting or finishing with KXK) that accounts for 41% of the for by this clone. A stretch of poly(A) was present at the 3‘ total lysine content.The relevance of this observation is end of the insert; however, no polyadenylation consensus unknown. sequence was identified. The regional expression of ESI-2.la mRNA was investiThe cDNA sequence data for ES1-2.la were used to deter- gated by subjecting chick tissues toreverse transcriptase PCR. mine potential similarities with more characterized molecules Total RNA was isolated from differentregions of the embryo within the GenEMBL data base through the Genetics Com- and used for cDNAsynthesis, followed by PCR amplification puter Group (Madison, WI). FASTA analysis showed that the in the presence of oligonucleotides to ES1-2.la (Fig. 3). The results indicated the presence of message for this protein in nucleotide and the amino acid sequences with the greatest homology to ES1-2.la were of little apparent significance the atrioventricular canal and outflow tract regions of the because: 1)the regions of identity were short, i.e. less than 4 heart, both of which undergo epithelial-mesenchymal transamino acids, 2) nucleotide homologies did not correlate to theformation events. In order to determine the cognatechicken protein encoded open reading frames for these molecules, and 3) regions of
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Q
S
T
D
Q
L
K
E
Y
I
S
L
L
E
A
Q
L
D
K
S
L
TTGCAGACAGCCAGCTCTGAACGCCAARACTACACAGAAG~AAGGCTM;AGACAGCTCTTATCAGAGTCCCAAGAGCAGC?T;GAGGCAGCGAAGACAGACGCAGAAGCAGAGC
---------+-.---..--+------...+~..--..~.+~~.~~~...+~~..~...~+
A
2281
K
V
AGGCAGAAGAAGACAGAGACCTGCAGGCAGCC~TGAAGCAG~TGAAGCAGCTGCAGCTMGGCTCCAGGAGAAAACGGACCAGCTCTn;TCC~GAAAGGGAAGCAGCAGAGC~C
S 2161
V
P
GCARATPGCTGGAGCTGGACAGCTn;AAGGGGAAACTGCAGGAAGCCAGCTCAGAGAACACAAGGCTTCTGGAGAGAATCAAATCCATCGAAGCTCTGCTGGAAGCAGGCCGGATGAGGG
Q 2041
P
V
"""".+""""~+"-"""+"""""~+".""..+""""~+""""~+"""."+"""".+""""~+""""~+""""~+
F 1921
G
E
TCCGCAAAACCCAGACCAGCTACAGGAGCCTGGTM;CAGATGCAGAAAAGGCCAAAGGGCAGCAACAGAGCATTGCTGAACTGCAGGCCAAGCTGCTGAGCTCTGAAACAGAGGTCAAAA
A 1801
D
K
----.-.--+---------+.-------+"------.--+-.""..-+-.-.--.--+.--.----.+--
E 1681
H
P
AGRAAAAGAGCTGGGAGATCAAAGCAGCAGCGTCAGAGAAGCAGA~AGCAGCTACAGAC?TCCCAARGAGAAATGGAGGCGACGCTGCAGAAGAGATTGGATGMGTMGCGACGAGC
A 1561
K
T
""""~+""""~+""-"""------+..""...+"".""+"".."~+"""."+"."""+""~"~.+""-"--+--"---"+
K 1441
Q
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AGGCTACCAGCCAGACAGAGAGCAAGCAAAACGCTGAGCTGGCCAAGTT.4CGGCAAGAATGCAACAAGCTGATGAAAGAGCTGTCTGAAAAATCAGAGGTGCTACAGCAAGAGGAGCAGC
R 1321
E
E
""""~+""~""+""--""+""""~+"""..~+".."."+..~...."+"".""+""""~+"""."+""-----+""--"-+
K 1201
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ACGAAACGCAGCAGCTCCAGGGAAAGATTCGGGCCCTGCAGGAACAGCTGGAGAACGGCCCCMCACACAGCTCGCTCGTCrrCAGCAGGAGAACTCCATCCTGAGGGATGCGCTCAACC
A 1081
H
V
CAGCTGAGAAGGCCAAGGCGGCAGCCAGTGAGAGCAAGCTGAAGGAACAGCTGGTCACCCGTGAACGGGAGATCACTGCTGTCCAGGCACGGATGCAGGCGAGCTACCAGGACCACGlTR """"~+""""~+-""""+"---""+""""~+"."""+"".""+"."""+""""-+""""-+"-"-"-+"----"-+
E 961
T
P
""""~+""""~+"-"""+""""-+""""~+""""~+""""~+""""~+""""-+""""-+""-----+-"----"+
A 841
P
S
AGGGTGACCCAGTTGCTGTCCTGAAGCGCCAGCTGGAAGAGAAGGAGAAGCAGCTCACTGCTGAGCAGGAGGATGCAGCTGCTGCCAGAAACAAGCTTCGGGAGTTGAGCAAGGAACTGG
G 721
L
G
AGACGCTGGTGrrCACCATCAGCAGCATGGCAT~AGCGMGGAGAGGCCCAGCAGCTCATCGAGATCCrrACAGAGAGAGCAGGCATCGTGCAGGACACCTGGCACACGGCTACGCAGA """".+""""~+-"""--+--------~+.""""+.""""+.".""~+..""...+""""~+"""."+""-"--+-"---"-+""-----+
T 601
K
D
S
Q
L
'
AAGGAGCTGGCCCTGGTCAGGCAGCAGCTGAGTGAGATGAAGATGAAGAGCCACGTGCAGGATGGAGMGTAGCGGGGTCACAAGCTGACC?~;GGCGATCCCACACCCTTTGAGTTGMAATGCAG
---------+-----.---+--"--"-+-----....~.+.~.~..~.~+~..~~
CTGGAGCAGAATGAGGCACTGATGGAGAAGGAGCMGAGCTGAGGCAAAAGCTGACGCGGGAGCTGGAGGAGGCACAAAGCTCAGCGTGCAGCTTGCAAGCAGAGCTGGAGAAGTTGAGA
- - - - - - - - - + - - - - - - - - - + - - - - - . - - + " - ~ - - . . . ~ . + . . . ~ ~ . ~ ~ . + . ~ . .
CTGGCTGAAAATGCAGCAGCTrrGGACATGGACATGGAGGAGGCCCAGCATCTCAAGGAllAGACTTG~~~ 2521
--~------+~~-~.~~..+-..~"-t---------f--.~...~~+..~~..~..+~
FIG. 2. Nucleotide and deduced amino acid sequences of ES1-2.la cDNA. Sequence for the entire2600-base insert of the ES1-2.la clone is shown starting from the 5' end. The longest open reading frame is in frame with the p-galactosidase promoter of pBluescript and extends for 2175 bases (725 amino acids); other reading frames exhibit multiple stop sites throughout the insert. A potentialN-glycosylation site (in boldface type) is encoded by bases 840-842. The first methionine is encoded by bases 507-509. A stop codon occurs at bases 21782180. The underlined sequences represent the location of sense (GGAGCAGCAAGTTACTCAGGAAGT)and antisense (GTTTCCGAGAGGACAGAACCTCCA) oligonucleotides used for PCR analysis (Fig. 3). Bases 1-839 of ES1-2.la cDNA were subcloned into pGEX-2T for antibody production (Fig. 4).
by ES1-2.la mRNA, a 20-kDa fragment of recombinant ES12.la protein was purified for the preparation of specific antibodies (Fig. 4A). N-terminal amino acid sequence analysis of this 20-kDa peptide confirmed its identitywith that predicted from the nucleotide sequence data (Fig. 4B;c.f. Fig. 2). Antibodies against this fragment of ES1-2.la protein detected a
single protein of 130 kDa in both the cell layer andthe conditioned medium from chicken cardiocyte cultures (Fig. 5). Therefore, the cognate protein for ES1-2.la subsequently was termed ES/130. Immunohistochemical analysis showed that ES/130 was expressed by cultured embryonic cardiocytes (Fig. 6), the
ES/130 in Developing Chick Heart
14408
A
1
2
3
4
kDa R
6997-
w m
41
259 *
31
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B I P G E X
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1
FIG. 3. Reversetranscriptase PCK analysis of E S l - 2 . l a ... V N R ~ G S P G C R N S A R G K I P... E expression in embryonic tissues. Primer-extendedcDNAfrom R S A ( 3 pg/reactionl of stage 11 heart( A 1. stage I 6 outflow tract ( R ) , FIG. 4. Isolation of an E S l - 2 . l a polypeptide expressed in and atrioventricular canal( I ) )were amplified hy PCR in the presence pGEX-2T. Panel A, samples from each step of purification were of ES1-2.la-specific primers (Fig 2, underlined sequences) resulting in a singleamplifiedproduct (259 hp). Lane C, amplifiedproduct from the ESl-Lla plasmid. Lana DSA standards (1-kilobase pair ladder).
E.
subjected toSDS-PAGE (13% acrylamidegels)andstainedwith Coomassie. IPTG-induced total bacterial lysate (lane 1 ) was subjected to glutathione-agarose affinity chromatography and the bound fraction (lane 2 ) treated with thrombin to release the 2.la' pol-ypeptide from glutathione S-transferase (/an? 3). The 2.la' pol.ypeptide was separated from glutathione S-transferase by ultrafiltration (lane 4 ). Panel H,amino acid sequence analysisof the purified ?.la' pol.ypeptide shows complete identity to the deduced amino acid sequence of ES1-2.la at the vector cloning site (larger lettars). The arrow head denotes the thrombin cleavage site between pGEX-derived glutathione S-transferase and the 2.la' polypeptide. The N terminus of the 2 . b ' polypeptide contains the vector-derived sequences GSP (pGI.:X) and GCRNSAR (pBluescript and linker sequences) due to the subcloningstrategy. The nucleicacidsequencein this region was ggatccccgggctgcaggaattcggcacgag.
conditioned medium of which elicits the transformation of AV endotheliumintomesenchyme(12).ThisES1antigen appeared to have a perinuclear distribution similar to the vacuolar system, e.g. endoplasmic reticulum. In some cells, staining appeared as accumulations or packages of granules indicative of secretory pathways. T h e in vivo expression of ES/130 was confirmed by the presence of a single band at 130 kDa in tissue homogenates (Fig. 7). Thisproteinwasobservedinembryonictissues harvested just prior to the onset of endothelial transformation. Most notably, ES/130 was detected in an EDTA extract of stage 16 hearts. Thisis significant because: 1) this was the immunogen for the original polyspecific ES1 antiserum and A B C D kDa of cushiontissue 2)thispreparationelicitstheformation F P mesenchyme (12). 200. In order to assess the potential involvement of ES/130 in 97the transformation of AV endothelium into mesenchyme an69 tibodies to ES/130 were tested for their ability to block this event in culture. When co-cultures of AV endothelium and myocardium were treated with anti-ES/130 IgG (1 gg/ml), 46mesenchyme formation was blocked and surface endothelial cells appeared rounded but did not detach from the collagen substratum (Fig. 8). After an additional 24 h, mesenchyme 30formation was observed (data not shown). The myocardium continued to beat throughout this period. Control cultures, i.e. thosewithouttreatmentorthatreceivedanexcess of 21.5preimmune rabbit IgG (250 pg/ml), transformed into mesento those obtained chyme within24 h. These results are similar 14.3previously with the polyspecific ES1 antibodies used toisolate the ES1-2.la clone; however, much higher concentrations of FIG. 5. Antibodies to ES1-2.la detecta 130-kDa protein in ES1 IgG were required to block mesenchyme formation (13). cultured cardiocytes. Both the conditioned medium and cell laver
from3-day-oldcardiocytecultures were subjected toSDS-PAGE (105 acrylamide) and immunohlotting with affinity-purified antihodies to 2.la' pol.ypeptide. Lane A , proteins from the cell laver stained T h e precise function of ES/130 in the morphogenesis of with Coomassie. Lanes R and C , anti-2.la' immunohlots of the cell cardiac enthelium is unclear. Our working hypothesis is that layerandconditionedmedium,respectively. Idant> D. preimmune cardiac mesenchyme formation is the result of several steps immunoblot of the cell layer sample. DISCUSSION
ES/130 in Developing Chick Heart
-
14409
FIG. 6. Immunohistochemical localization of ES1-2.la in cardiocyte cultures. Day 3 cardioc.yte cultures were stained with affinitypurified antibodies to the 2.la’ polypeptide. Panel A, phase-contrast image. Panels &I), fluorescent images. Note perinuclear (panel (’) and secretory-type granular ( p a n e l D ) localizations of ES1-2.la. Magnification, X 82 ( A and R ) and x 820 ( C and D ) .
down-regulation of theneural cell adhesion molecule (NCAM) (3,6),Golgi polarization (ll), andincreased expression kDa of substrate adhesion molecules (5), TGF-@ (33), and uroki200nase-type plasminogen activator (4),before subsequent invasion of the ECM between the endothelium andmyocardium. 97I t seems likely that the complete process of endothelial trans69formation is not an all-or-none phenomenon, since it ispossible to affect certain characteristics without the formation of 46mesenchyme. This is supported by the observation that hyaluronidase treatmentof heart EDTA extract negates its ability to induce mesenchyme formation, yet the endothelium cells 30separate from one another, hypertrophy, and have polarized Golgi (11). Additionally, in a recentreport by Pottsand Runyan (34), it was shown that in the presence of TGF-@3 21.5antisense oligonucleotides, cardiac endothelial cells undergo typical morphological changes in culture, but again no mes14.3enchyme was formed. In the present study, antibodies to ES/ FIG. 7. Antibodies to E S 1 - 2 . l a d e t e c t a 130-kDa protein in 130 inhibited the epithelial-mesenchymal transformation of the EDTA extract of the embryonic hearts. Samples were suh- AV endotheliuminculture.Sincetheendothelial cells in jected to SDS-PAGE(10% acrylamide) and immunohlotting with these cultures separated from one another but remained ataffinity-purified antibodies to 2.la’polypeptide. Lunes A and B, total tached to the substratum and non-invasive, ES/130 may be stage 16 embryo stained with Coomassie and anti-e.la’, respectively. in up-regulating the Lane C, immunoblot of EDTA extract from stage 16 heart tissue. involved, either directly or indirectly, Lane D,immunoblot of heart tissue after EDTA extraction. expression of substrate adhesion molecules. Recently, c-fos has been implicated asa potential regulator that require multiple signals. During this morphogenetic proc-of epithelial-mesenchymal transformation of mammary epimay link a short ess endothelium exhibits many morphological and molecular thelium (36). It is thought that this protein characteristics, including cellular hypertrophy (2), expressionterm stimulus to a pronounced change in cell morphology, influx (32), loss of cell-cell contacts via a such as epithelial-mesenchymaltransformation.Mammary of Hox-7 (31),ca2+
A
,
A
”
-
6
C
D
14410
ES/130 in Developing Chick Heart ES/130 and scatter factor. Inaddition, hepatocyte growth factor does not elicit any of the characteristics of cardiac endothelial transformationin culture.2 Wehave preliminary immunohistochemical and in situ hybridization data which indicate the presence of ES/130 mRNA and protein in limb bud ectoderm, dorsal ectoderm overlying the neural tube, notochord, and floor plate of the neural tube.' In these embryonic sites, inductive interactions between neighboring tissues have been postulated (28-30). It is interesting to note that only those tissues thatexpress ES/ 130 can elicit the formation of mesenchyme from AV endothelium in culture, e.g. notochord and limb ectoderm uersw skeletal myoblasts.2 Immunohistochemical studies have shown that a complex of ES1 antigens islocalized as extracellular, multicomponent particulates in several areas of the chick embryo undergoing epithelial-mesenchymal interaction (3,13,35). Although some of the morphogenetic events in these areas appear similar to AV endothelial development, e.g. neural crest formation, the correlation a t other ES1-positive sites is not as obvious, e.g. ectodermal ECM in limb morphogenesis. These observations may result in part from a differential distribution of individual ES1 antigens, which either elicit separate events or modify the function of the inducing agent. Therefore, itis important to generate specific probes to additional ES1 antigens and examine their co-distribution with ES/130, as well as assess their biological function. In conclusion, ES/130 appears toplay an important role in the transformation of early cardiac endothelium intocushion tissue and may also be involved in other areas of inductive interaction. The distribution of this protein throughout the molecular charembryo and its potential relationship to other acteristics of endothelial differentiation are currently being investigated.
FIG. 8. The transformation of AV endothelium into mesenchyme in culture is blocked by antibodies to ES/130.Mesenchyme formation from AV explants was ohserved by 48 h in both untreated cultures (panel A ) and those that received non-immune IgG (250 pglml; panel H ) . Cultures treated with anti-ES/lBO (1 pg/ ml; panel C') did not form mesenchyme. The extent of proliferation of the surface epithelium was similar to controls.Although these cells appeared to lose cell-cellcontactsthey remained attached to the suhstratum. After an additional 24 h, mesenchymal cells were ohserved within the collagen gel. In all cases the myocardium remained heating throughout the entire culture period. Magnification, X 29'i.
Acknou,leQments-We thank Sue Tjepkema-Burrows and Carolyn Snyder for preparing the figures, Matt Mintz and Nanette Paul for technical assistance, Dr. Liane Mende-Mueller and Rrady Stoner at the MCW protein and nucleic acid facility, and Drs. .Joe Rarbieri and Rick Sabina for critical comments. REFERENCES 1. Davis, C. L. (1924) Anat. Rec. 27,201-202 2. Markwald, R. R.. Fitzharris, T.I'.. and Adams-Smith, W . N. (1975) I k c . Hiol. 4 2 , 160-180 3. Miaatvedt, C. H., and Markwald, R. R. (1989) Dcu. Aid. 119.59-67 4. Mi-(hire. P. G., and Orkin, R. W . (1992) Dro. I l w . 1 9 3 , 24-33 5. Funderlmrg, F. M., and Markwald. H. R. (1986) J . ( ' d l Hiol. 1 0 3 , 2475"87
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epithelial cells transfected with c-foslestrogen receptor fusion constructs responded to estrogen treatment by transforming from an epithelialmorphology into an invasive, mesenchymal population, with concomitant loss of epithelial-specific markers and theexpression of those characteristicof mesenchyme. Should c-fos be expressed by cushion tissue in the earlychick heart, it would be interesting to investigate its dependence upon ES/130 treatment of cardiac endothelium. The lack of apparent homology to known proteins indicates that ES/130 is a novel protein. However, ES/130 is not the only protein to be implicated asamediator of epithelialmesenchymal transformation. Scatter factor, a 92-kDa heparin-binding glycoprotein with significant homology to hepatocyte growth factor, was isolated from MRC5 human fibroblasts and can affect the epithelial-mesenchymal transformation of Madin-Darby canine kidney epithelial cells and generatesupernumerary axial structures in chick embryos (37, 38). To date we have not found any homology between
348455
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' M. Rezaee and E. L. Krug, unpublished observations.
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ES/130 in Develc3ping Chick Heart 21. Devereux, J., Haeberli, P., and Smithies, 0.(1984) Nucleic Acids Res. 1 2 , 387-395 22. Kawasaki, E. S. (1990) in PCR Protocols: A Guide to Methods and A pli cations (Innis, M.A., Gelfand, D. H., Sninsky, J. J., and White, l! J, eds) pp. 21-27, Academic Press, San Diego 23. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1989) Molecular C h i n A Laboratory Manual, Cold Sprmg HarborLaboratory, Cold Spring Hartor, NV
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31. Robert, B., Sassoon, D., Jacq, B., Gehring, W., and Buckingham, M. (1989) EMBO J . 8.91-100 32. Runyan, R. B., Potts, J. D., Sharma, R.V., Loeber, C.P., Chiang, J. J., Bhalla, R. C.(1990) Cell Regul. 1,301-313 33. Akhurst. R. J.. Lehnert. S. A,, Faissner,. A,.. and Duffle, E. (1990) Deuelopment ios, 645-656 34. Potts, J. D., Dagle, J. M., Walder, J. A,, Weeks, D. L., and Runyan, R. B. (1991) Proc. Natl. A d . Sci. U. S. A. 88. 1516-1520 35. Mj’aatvedt, C. H , &era, R. C., and Maikwald, R. R. (1987) Deu.Biol. 119,59-67 36. Reichmann, E., Schwarz, H., Deiner, E. M., Leitner, I., Eilers, M., Berger, J., Busslinger, M., and Beug, H.(1992) Cell 71,1103-1116 37. Stern, C. D.,Ireland, G. W.,Herrick, S. E., Gherardi, E., Gray, J., Perryman, M., and Stoker,M. (1990) Deuelopment 110,1271-1284 38. Weidner, K. M., Behrens, J., Vandekerckhove, J., and Birchmeier, W. (1990) J. Cell Bid. 1 1 1,2097-2108
