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1274
Bovine Angiotensin Converting Enzyme cDNA
Cloning and Regulation
Increased Expression During Endothelial Cell Growth Arrest
Shaw-Yung Shai, Robert S. Fishel, Brian M. Martin, Bradford C. Berk, and Kenneth E. Bernstein
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Angiotensin converting enzyme (ACE) is a zinc-containing dipeptidase that converts angiotensin I to
angiotensin II, a powerful vasoconstrictor and smooth muscle growth factor. ACE activity has been shown
to be dynamically regulated by hormones, ACE inhibitors, and endothelial cell growth state. To study how
ACE expression is regulated, we isolated and sequenced the bovine ACE gene using both ACE-specific
cDNA and genomic clones. Bovine ACE cDNA encodes a single polypeptide of 1,306 residues with a
molecular mass of 150 kd. Bovine ACE is approximately 80%'o homologous to that of other species. It
contains two homologous domains of equal size. Alignment of ACE sequences from bovine, human, mouse,
and rabbit reveals that during evolution both domains have been highly conserved. We used the bovine
ACE cDNA to study regulation of ACE gene expression during density-dependent growth arrest. As
endothelial cells became growth-arrested (6 days after confluence), there was a 12-fold increase in ACE
activity and a 90%o decrease in DNA synthesis. Immunocytochemically detectable ACE markedly increased
in growth-arrested cells. The increase in ACE was due to increased ACE gene expression, as assayed by
RNase protection, which showed a 20-fold increase in ACE-specific mRNA. The present study shows that
bovine ACE is highly regulated by endothelial cell growth state at the level of protein and mRNA
expression. Such dynamic regulation may have important consequences for angiotensin II production
during endothelial cell proliferation after arterial injury. (Circulation Research 1992;70:1274-1281)
KEY WoRDs * cDNA * cloning * DNA * RNase protection
ngiotensin converting enzyme (ACE) is a zinccontaining dicarboxypeptidase that converts
angiotensin I to the potent vasoconstrictor angiotensin ll.1-3 ACE is produced by several tissue types.
However, it is the production of this enzyme by vascular
endothelium that is believed to be most important for
systemic blood pressure regulation. ACE is localized on
the luminal surface of endothelial cells as an extracellular protein, well situated to hydrolyze intravascular
angiotensin I.4 The resultant angiotensin II is in proximity to vascular smooth muscle cells, one of the critical
target tissues for this peptide. Thus, a major axisregulating vascular tone is vascular smooth muscle
vasoconstriction in response to endothelial production
of angiotensin II.
A
From the Departments of Pathology (S.-Y.S., K.E.B.) and
Medicine (R.S.F., B.C.B.), Emory University School of Medicine,
Atlanta. Ga., and the Section on Molecular Neurogenetics
(B.M.M.), Clinical Neuroscience Branch, National Institute of
Mental Health, Alcohol, Drug Abuse, and Mental Health Administration, Bethesda, Md.
Supported by an Emory Georgia Tech research award (B.C.B.),
a Grant-in-Aid from the American Heart Association, Georgia
Affiliate (K.E.B.), and Public Health Service grant DK-39777
(K.E.B.). R.S.F. was supported by National Research Service
Award from the National Institutes of Health. K.E.B. is an
Established Investigator of the American Heart Association.
Address for correspondence: Dr. Kenneth E. Bernstein, Department of Pathology, Emory University School of Medicine,
Atlanta, GA 30322.
Received October 22, 1991; accepted February 24, 1992.
Endothelial cell expression of ACE has been investigated in several different species using cultured endothelial cells. These studies show that several different
stimuli, including steroids and other hormones, cell
passage number, intracellular calcium, and ACE inhibitors, modulate endothelial ACE expression.5-9 One of
the most interesting observations is that actively proliferating endothelial cells contain very little ACE; high
levels of the enzyme are observed only in endothelium
at confluence when the cells are relatively nonproliferative.10,11 To investigate the biochemical regulation of
ACE expression as a function of bovine endothelial cell
proliferation, we have undertaken the cloning of bovine
ACE. Here we demonstrate that cultured bovine endothelial cells markedly upregulate ACE expression when
they reach a nonproliferative state and that this is
associated with a parallel increase in the amount of
bovine ACE mRNA.
Materials and Methods
A bovine lung cDNA library cloned in lambda gtlO
was purchased from Clontech Laboratories, Inc. A
bovine genomic library in lambda DASH was also
purchased from this company. DNA sequencing was
performed with double-stranded plasmid DNA or portions of DNA cloned into M13 using Sequenase' and
other reagents purchased from United States Biochemical Corp., Cleveland, Ohio.12,13 Sequence information
was analyzed using GENEPRO (Riverside Scientific, Bainbridge Island, Wash.) and PC/GENE (IntelliGenetics,
Mountain View, Calif.) software.
Shai et al Bovine ACE cDNA Cloning and Expression
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Bovine aortic endothelial cells were isolated from a
young calf as previously described.14 The cells have the
following characteristics typical of endothelial cells:
growth in monolayers with a "cobblestone" appearance
at confluence, uptake of 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (Dil-AcLDL),15 and expression of ACE. The cells used for the
studies reported in this paper were passage numbers
7-13. For cell confluence studies, cells were plated at
1x106 cells per plate on a 10-cm culture plate in
Dulbecco's modified Eagle's medium supplemented
with 10% fetal calf serum and penicillin/streptomycin at
100 units/ml and 100 ,ug/ml, respectively. Culture medium was changed every 2 days. Confluence was assessed visually and was defined as complete coverage of
the tissue-culture plastic by the cell monolayer.
To determine ACE enzyme levels, endothelial cells
were washed twice with 10 ml warm phosphate-buffered
saline (PBS) to remove any serum components. Cells
were then scraped into 5 ml ACE assay buffer (50 mM
HEPES [pH 7.5] and 100 mM NaCl). The ACE assay
was performed as described by Ryan and colleagues.'6"17
Briefly, 1 ml cell suspension (-0.5 mg protein) was
dispensed into a tube, and Triton-X 100 was added to a
final concentration of 0.05%. This was prewarmed to
37°C, and proteins were solubilized for 5 minutes. To
start the assay, 50 g1 assay buffer containing 1 gCi
['H]benzoyl-phenylalanine-alanine-proline (Ventrex
Laboratories, Portland, Me.) was added. Aliquots (50
,ul) were removed at 15 and 30 minutes and acidified to
prevent further reaction. The aliquots were then extracted with Ventrex scintillation cocktail No. 2, and the
hydrolyzed ['H]benzoyl-phenylalanine was measured by
liquid scintillation spectrometry. For determination of
specific ACE activity, parallel incubations were performed containing either 1 ,uM lisinopril (an ACE
inhibitor), a blank (buffer only), or a positive control (50
gug bovine kidney extract). ACE activity was defined as
lisinopril-inhibitable enzyme activity. Enzyme activity
was correlated to radioactivity within the scintillation
cocktail using a formula based on Michaelis-Menten
kinetics as described in the Ventrex protocol. Protein
was determined by the method of Bradford,18 and ACE
enzyme activity was expressed as units per microgram
protein.
To relate the expression of ACE to cell confluence,
bovine endothelial cells were plated on 35-mm tissue
culture plates. These plates were then placed at a 300
angle, which caused the cells to plate in the lower half of
the dish. Cells grew for 3 days, and then the dish was
placed in a horizontal position. Cell proliferation occurred as the cells migrated into the section of the dish
previously uncovered by media. The plated cells were
fixed for 12 minutes in Bouin's solution (70% saturated
picric acid solution, 25% formaldehyde solution, and
5% glacial acetic acid). They were then washed twice in
70% ethanol and twice in PBS. The cells were first
incubated with 1% sheep sera for 15 minutes and then
washed twice with PBS. An antibody dilution of 1: 200
of a rabbit anti-mouse ACE antisera was applied to the
dishes for 1 hour at room temperature.19 In a control
plate, a 1:100 dilution of rabbit preimmune sera was
used. The plates were washed with PBS and reacted
with biotinylated anti-rabbit immunoglobulins (LINK
reagent, BioGenex Laboratories, San Ramon, Calif.)
1275
for 30 minutes at room temperature. The plates were
washed with PBS, and peroxidase-conjugated streptavidin was added (LABEL reagent, BioGenex Laboratories) for 30 minutes at room temperature. The plates
were washed with PBS, and a solution of 3-amino-9ethylcarbazole (Signet Laboratories) was added for 12
minutes.
To assay DNA synthesis, ['H]thymidine incorporation was measured.20 Endothelial cells were labeled in
the presence of 10% serum with 1 uCi/ml [methyl3H]thymidine (20 Ci/mmol) for 24 hours. After labeling,
cells were washed with cold saline, trypsinized, and
collected by centrifugation (lSOg for 5 minutes). The
cell pellet was suspended in cold 10% trichloroacetic
acid and agitated vigorously to lyse the cells. Nucleic
acids were collected on a glass fiber filter (GF/F). After
washing with cold 5% trichloroacetic acid and 70%
ethanol, respectively, the filter was dried and counted in
a liquid scintillation counter (model LS5000 TD, Beckman Instruments, Inc., Fullerton, Calif.).
To prepare total RNA, endothelial cells were washed
with PBS (pH 7.4). Two milliliters of 4.4 M guanidine
isothiocynate was added to each 10-cm plate, and the
cells were scraped into this buffer.21 The cell lysate was
then passed 10 times through a 20-gauge needle. RNA
was pelleted through a two-step CsCl gradient (1 ml of
a 40% solution of CsCl in 10 mM Tris [pH 7.5], 1 mM
EDTA (TE) layered over 3 ml of 5.7 M CsCI in TE) in
an SW41 rotor (35,000 rpm for 16 hours).22 The RNA
pellet was dissolved in water, extracted with phenolchloroform, and precipitated.
A 470-base antisense riboprobe was synthesized from
cDNA pBLA15.23 Plasmid DNA was digested with Xho
I, and radioactive RNA was synthesized using T7 RNA
polymerase (Stratagene Inc., La Jolla, Calif.) under
conditions supplied by the manufacturer. [a-'2P]UTP
(800 Ci/mmol) was purchased from Amersham Corp.,
Arlington Heights, Ill. Other reagents were obtained
from Stratagene. The template DNA was then digested
with 15 gg RNase-free DNase I (GIBCO BRL, Gaith+1
264
265
432
BG2
eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeI4179
II
BTAcK
300
1330
BLA13
384
BLIS
2718
i~~~~~~~~~~~~~~~~~~~~~~~~~~~~
1318
3690
BL&S
2830
4179
BLA12 .3
500bp
FIGURE 1. Schematic diagram of bovine angiotensin converting enzyme cDNA. BLACE is a schematic representing the
sequence of bovine angiotensin converting enzyme. It is the
composite of cDNA clones BLAJ3, BLA15, BLA5, and
BLA12.3. The 5' end of the bovine gene, positions 1-432, is
derived from genomic clone BG2. Here, exons are indicated
by the hatched boxes, and introns are indicated by a line.
Numbers over the individual boxes indicate the 5' and 3'
positions of individual cDNA clones or genomic exons.
Circulation Research Vol 70, No 6 June 1992
1276
1
ATGGGGGCCGCGTCGGGTCGCCGGTCGCCGCCGCTGCTGTGGCGTTGCTGUCTGCTUGC
N G A A S GC R SFP P L L L P L L L L L
CTGCCGCCGCCGCCCGTGATCCTGGAGCTGGACCCCGCGTTGCAGCCGGGGAACTTTCCC
L P
P
P
P V
L D P A L Q
ILL
A
D
E A G A Q I
F A A S
F N S
681
T A A
S
W A H D
T N
I
]180
721
300
761
R
L Q E E A A L L S Q E F S
P V WQ N
F T
D
I
ATCGGGGCGGTGCGCACCCTGGGCCCCGCCAACCTGGACCTAGAGAAGCGGCAGAAGTAC
121
I
G A V R T
S
L G
L L S N N S
T A K V C
420
161
K
T A P
C W
S L D
P E L T N I
L A
Y T L L L Y A W E G W H N A A G
S
I
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540
841
660
881
780
921
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P
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F T A L
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F
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R L Y Q Q L E
D R Y I
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P
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281
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L G
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S
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321
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L G
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961
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Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017
H
L E K P
S
D
G R E V V
G
H V Q Y Y L Q
TACAAGGGCCAGCACGTCTCCCTGCGCCGAGGCGCCAACCCCGGCTTCCACGAGGCCATC
401
Y
K G Q H V
S
L R R G A N P
G
F H
E A
1260
I
GGCGATGTGCTGGCCCTCTCCGTCTCCACTCCTGCACACCTGCACAACATCGGCCTGCTG
G D V L A L S V S
441
481
T P A H L H K
I
G
L L
GACCAGGTCACCAATGACACAGAGAGTGACATCAACTACTTGTTAAAGATGGCACTGGAA 1380
D Q V T N D T E S D I N Y L L K N A L E
AAAATTGCCTTCCTCCCTTTGGCTACTTGGTGGACCAGTGGCGTTGGGGGGTCTTTACT
R W CG V F S
K I A F L P F G Y L V D Q
GCCCGAACCCGCCCTTGCCCGCTACAACTATGACTGGTGCTATCTTCGAACCAAGTATCAG 1500
G
R T R P
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f
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1041
521
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561
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601
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2580
L H A Y V R R A L
I
N L E C
P
I
P A H L L
E A
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F
K A N I K Q CG
F T
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L G
L L
T
P M
P
L RN
P P
K L E K P T D G R E V V
CN
E
F
A S
W D F F N C K D F R I K Q C T S V N N
GAGGACCTGGTGGTGGCCCACCACCAAATGGGCCACATACAGTACTTCATGCAGTACAAG
E D L V V A H H E H G H I Q Y F K Q Y K
A
D
L
P V
T
G
D G
G Y
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G A N
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3060
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A
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N F L N K N
L L S
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3180
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GCCTTCATCCCCTTCAGCTTCCTCGTCGACCAGTGGCCGT0CACGGTGCTTTATGGAACT
O
I
F S F
L V
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V R V F D
G
1201
TTCAAGCCGCTGGTGGACTGGCTCGTGACCGAGAACGGGCGCCAC0 GCACAAGCTCCCC
F K P L V D W L V T E N G R H C E K L C
TGGCCTCAGTACAACTGGACGCCAAACTCGGCTCGCCCTGGCGGGCCCTTCGTGG=CCCC
1241
G
Y N W T P N S A R P GC
P F V G S
F L G L N L E E Q Q A
R V C Q W
3660
R V N
CTCCTGCTCTTCCTGGGCCTCGCCCTGCTGGTGGCCACGCTGGGCCTCACCCAGCGGCTC
V
L
L
F L G V A L L V A T L
C L T
Q R L
TTCAGCATCCGCCACCACAGCCTCCGCGGGCCCCACCGCGGCCCCCAGTTTGGCTCCGAG 3900
1860
1281
S I R H H S L R C P
GTGGAGCTGAGACACTCCTGA
V E L R H S *
F
H R C P Q F
G S
E
3921
3981
4041
4101
4161
E Y D R R S
CAGGTCGTGTGGAACGATATTGCCGAGGCCAACTGGAACTACACACCGACATCAGCACG
A N W N Y S T D I S T
Q( VV W N E Y A
AACMACACATCCCMACCACACCGTCAACTAT
GACAACAGCAACT11 WCAT1 ACa
P
GGGAACATGTGGGCACAGACCGCCTCCAACATCTATCACTTCGTGCCACCCTTCCCTTCA 2700
G N M W A Q S V S N I Y D L V A P F P S
GCCCCCAAGATGGATGCCACGGAGGCCATGATAAAGCAGGGCTGCACACCCCTAAGGATC
GACCTGGTGTCCGACGAGGACGAGGCCCGCAAGTTICTCCAGGAATACGACCCCAGATCC 1980
641
E L Q
CACCGCCACTACGGGCCCGACGTCATCAACCTGGAGGGCCCCATCCCAGCCCACCTGCTC
W P Q
D N L D A R P L L
AGCTACTTCCAGCCGGTCACACAGTGGCTCGAGGAGCAGAACCAGCAGAACGGCGAGCTC
S Y F Q P V T Q W L E E Q N Q Q N C E V
CTGGGCTGGCCAGAGTATCAGTGGCGCCCACCGATGCCCGACAATTACCCGGAGGGCATT
f P P H P D N Y P E G I
L G V P E Y Q
R
2460
GAAGCCATGCGGCTGATCACGGGCCAGTCCAACATGTCTGCCTCCGCCATGATGACCTAC
E A KR L I T G Q S N K S A S A K H T Y
TGGCAGGAGCTGCTIAAGCACATGGCTGGCTCAGACAACCTGGACGCTACCGCTGCTC
E V L K D
I L P
E L T N K A A R L N G Y Q D
1161 K E A G K L L A D A K K L C F S Q P V P
Q
V Q
F P K Y V
GGCCGCGTCAACTTCCTGGGCCTGAACCTGGAGGAGCAGCAGGCCCGCGT GCCAGTGG 3780
I Y
CAGTCCACCCAGGCGGGGGCCAAGCTCCGCCGCCTCGCAGGCACCCTCCTCACGGCCC
S T Q A G A K L R A L L Q A G S S R P
R N Y
GTCACCAGGGAGAACTACAACCAGGCAGTGTGGAGCCTCAGGCTGAAGTACCAGGGTGTC 3300
TGTCCCCCACTGGCCAGATCTCAAGATGAIGACCCAGGGGCCAAGTTCCACATTCCT
C P P L A R S Q D D F D P C A K F H I P
CCAAGTGTGCCTTACCTCAcCTACTTCCTCAGCTTTCTCATCCACTTCCAGTICCACGAC 3420
1121 A S V P Y V R Y F V S F V I Q F Q F H Q
GCGCTCTCTCAGGCAGCGGGCCACCAGGGCCCCCTCCACAACGTGTCACATCTACCACTCC
A L C Q AA G H Q C P L H K C D I Y Q S
AGGACGCCGGGCMGCTCCTGGCGGATGCCATGAAGCTGGGCTTCACTCAGCCGTGGCCC 3540
GGGATCTGTCCTCCGGTTCTCCGAAATGAAACCCACTTCGACGCTGGAGCCAAGTTTCAC
G
5
1081 V T R E N Y N Q E V V S L R L K Y Q G V
L R T K Y Q
GTCCCAAATGTGACCCCGTATATCAGGTACTTCGTGAGCTTTCTCCTGCAGTTCCAGTTC
V P N V T P Y I R Y F V S F V L Q F Q F
CACCAAGCGCTGTGCAAGGAGGCAGGCCACCAAGGCCCCCTGCACCAGTGTGACATCTAC
C H E N
GGGCACGGCGGCTACGAGGAGGATATCAACTTTCTGATGAAGATGGCGCTCGAGAAGATT
A S A W D F Y N R K D F R I K Q C T
ACTATGGACCAGCTGTCCACGGTGCACCACGAGATGGGCCACGTGCAGTACTACCTTCAG
T V NH H E
L M A T
GTGCTGGCCCTCTCAGTCTCCACCCCCACGCACCTGCACAAGATCAACCTGCTGAGCACT
R V
T H D Q L S
D L T N
V L A L S V S T P T H L H K I N
C H
GCGTCCCCCTGGGACTTCTACAACAGGAAAGACTTCAGGATCAAGCAGTGCACGCGGGTC 1140
361
P
GACTTGCCCGTGACCTTCCGGGAGCGCGCCAACCCCCCTTTCCGAGGCCATTGCCCAC
1001
P P
GAGTTCTGGGCGGAATCCATGCTGGACAAGCCAAGCGACGGGCGGGAGGTCGTGTCCCAC
E F W A E S
L R L E
GCCTGGGACTTCTTCAAcCCCAACGGCACAGATCAAGCAGTCACCTCGGTGAACATG 2940
900
CCCGACAAGCCCAATCTTGATGTTACCGATGTTATGGTGCAGAAGGGCTGGAACGCCACA
P D K P N L D V T D V N V Q K G W N A T
CACATGTTCCGGGTGGCGGAGGAGTTCTTCACCTCCCTGGGGCTCTTCCCCATGCCACCC 1020
F F T
L L D M E T V Y S V A S V
W N K S
P A H
T V V
C
TGGAACAAGTCGATGCTGGAGAAGCCGACTGATGGGCGCGGAAGTCTCTCCACGCCTCC
P L Y L N L H A Y V R R
CTGCTGGGGAACATGTGGGCCCAGAGCTGGGAGAACATCTACGACACGGTGGTGCCCTTC
2220
TTTAAGGAAGCAGACAATTCTTCACCTCCCTGGGGCTGCTGCCCATGCCCCCTGAATTC 2820
GCACTGCACCGCCGATATGGGGACAGATACATCAACCTCAGGGGACCCATCCCCGCTCAC
A L H R R Y G
Y
A
L
GAACGCCTCTACCAGCAGCTGGAGCCCCTCTACCTGAACCTCCATGCCTACGTCCGGCGC
241
T
H R H Y C
TCAGACACAGGGGCCTACTGGCGCTCCTGCTATGACTCTCCCACCTTCACGGAGGATCTG
S
G
G D S W R S M Y E M F F L E E E L E Q
CTGTTCCAAGAGCTGCAGCCGCTCTACCTGAACCTGCACGCCTACGTGCGCCGGGCCCTG
P L
AAGCCCCTATACCAGGACTTCACTGCCCTCAGCAACGAGGCCTACAAGCAGGATGGCTTC
201
K R X
GGCGGGGACTCCTGGAGGTCCATGTACGAGATGCCCTTCCTAGAGCAGGAGCTGGAGCAG
AGCTACACCCTGCTGCTGTATGCCTGGGAGGGCTGGCACAACGCCGCGGGCATCCCACTG
S
T V K Y
G
F P N
AAGACTGCCCCCTGCTGGTCCCTGGACCCAGAGCTCACCAATATCCTGGCTTCCTCGCGA
N E K N L Q H A N H
F D V T N F Q N A T N
TACTTCCCCAAATACGTGGAGCTCACCAACAAGGCCGCCAGGCTCAATGGCTACCAGGAT
801
P A N L D L E K R Q K Y
R I Y S
L
K
Q D L A V A W K S W R D K V G R S
AACTCTCTGCTAAGCAACATGAGCAGGATTTACTCCACTGCCAAGGTCTGCTTCCCCAAC
N
S
CAGGACCTGGCCTGGGCGTGGAAGACCTGGCGCGATAAAGTGGGGCGGTCCATCCTACCC
E A W G Q K
P T L L R
L
GGCACCTGCCTGCGGCTCGAGCCTGATCTGACCAATCTGATGGCCACATCCCGGAATTAT 2340
GCCAAGGATCTGTTCGACCCGGTCTGGCAGAACTTTACCGACCCCACGCTGCTGCGCATC
A K D L F D
K
ATAAAGAAGATTCAGGATCTAGAGCGGGCAGCACTGCCCACCAAGCACCTGGAAGAGTAT
I K K I Q D L E R A A L P T K E L E E Y
N Q I
E N A
CGGCTCCAGGAGGAAGCAGCCTTGCTCAGCCAGGACTTTCAGAGGCCTGGGGCCAGAAG
81
S
T W A
AACCAGATCCTGCTGGACATGGAGACCGTTrACAGCGTGGCCTCTCTGTGCCACGAAAAT
S A E Q V
T E
N
GGCACCTGGGCCAGCAAGTTTGACGTGACCAACTTCCAGAATGCCACCATGAAGCGGATG
CTGTTCCAGAGCACGGCCGCCAGCTGGGCGCACCACACCAACATCACCGAGGAGAACGCG
L F Q S
D
G
P G N F P
GCCGACGAGGCCGGGGCGCAGATCTTCGCAGCCAGCTTCAACTCGAGCGCCGACCAGGTG
41
60
AAA
2100
FIGURE 2. The cDNA sequence of bovine angiotensin converting enzyme and the deduced amino acid sequence. The A of the
initiating methionine is +1, and the A of the termination codon TGA is +3921. The amino acid sequence is indicated under the
nucleotide sequence. The numbering of the nucleotide sequence is shown on the right side of the sequence, and that for the amino
acid sequence is on the left. The glutamic acid at position 29 (boldface E on second line of amino acid sequence) is the N-terminal
residue ofpurified bovine renal angiotensin converting enzyme and results from the removal of the hydrophobic N-terminal leader
sequence.28
ersburg, Md.) for 60 minutes at 37°C. The riboprobe was
extracted with phenol-chloroform and precipitated. Endothelial RNA (20 gg) was hybridized in a volume of 30
,.t with 2-3 x 106 cpm riboprobe. The hybridization
solution contained 80% formamide, 40 mM PIPES (pH
6.4), and 400 mM sodium chloride.24 The riboprobe was
hybridized with the RNA overnight at 45°C. RNase
digestion was performed in a volume of 350 gl containing 40 mg/ml RNase A (Sigma Chemical Co., St. Louis,
Mo.) and 0.14 units/gl RNase Ti (GIBCO BRL).
Digestion was for 60 minutes at 30°C. After deproteination and precipitation, the reaction products were
resolved on a 5% polyacrylamide DNA-sequencing gel.
ACE mRNA is expected to protect 407 bases of the
470-base riboprobe.
To control for conditions of RNA hybridization and
digestion, a 170-base Hinfl-HindIII fragment of bovine
,3-actin cDNA served as the template for the synthesis
of a /3-actin riboprobe.25 Thus, each experimental hybridization contained two riboprobes, that for bovine
ACE and 5 x 105 counts for bovine actin RNA.
Results
One million cDNA clones were screened by plaque
hybridization with mouse ACE cDNA pACE.31 and
pACE.11.26,27 Four positive cDNA clones were plaque
purified, and the cDNA sequence was determined (Figures 1 and 2). Sequence comparison with mouse ACE
indicated that none of these cDNA encoded the 5'
Shai et al Bovine ACE cDNA Cloning and Expression
BOVINE
cDfl
BOVINE
HUMAN
MOUSE
RABBIT
86
80
NA
83
NA
HUMAN
84
MOUSE
82
82
RABBIT
82
81
NA
84
FIGURE 3. Chart comparing the amino acid (AA) and
nucleotide sequences of bovine, human, mouse, and rabbit
angiotensin converting enzyme. Computer analysis was used
to determine the AA and nucleotide homologies among the
published angiotensin converting enzyme sequences. The
cDNA homologies are listed along the top portion of the
figure;AA conservation is listed along the bottom portion. NA
indicates a comparison not performed.
Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017
portion of the bovine transcript. To identify the 5' most
coding portion of the gene, a bovine genomic library was
screened with a 209-base pair Sac II fragment from the
5' end of the bovine cDNA, pBLA15. The genomic
clone BG2 was isolated and a 1,500-bp portion of this
clone was sequenced. Figure 1 shows the exon-intron
boundaries for the first two exons based on sequence
analysis of the genomic clone. Figure 2 shows the
composite cDNA sequence of bovine ACE based on the
cDNA and genomic DNA sequences. The sequence
1
-
.9
c
.7
am
.5
0
0
c
.4
a)
.3
.2
.1
-1
0 -1
1
.
209
4600
608
ame
lm
1211
Amino Acid Position
FIGURE 4. Graph showing sequence identity of bovine,
mouse, human, and rabbit angiotensin converting enzyme.
The PC/GENE program CLUSTAL was used to align the amino
acid sequences of mouse, human, rabbit, and bovine angiotensin converting enzyme. A consensus was then generated.
Positions in which there was amino acid identity among the
four sequences were marked. To quantitate sequence conservation, a window of 40 was centered at every position of the
consensus, and the number of marked positions was counted.
The y axis indicates the fraction of marked positions; the x axis
is the position along the consensus. The reduction of sequence
homology centered at position 140 is due to a small region of
nonhomologous sequence within rabbit angiotensin converting
enzyme. Arrows indicate the potential zinc-binding sites in
both the N-terminal and the C-terminal domains of angiotensin converting enzyme.
1277
predicts a protein of 1,306 amino acids with a postulated
molecular mass of 150 kd. The composite cDNA exactly
predicts the amino acid sequence determined experimentally for purified bovine ACE and indicates that
bovine ACE is translated with a 28-amino acid leader
sequence.28
cDNA-encoding ACE has previously been obtained
for mouse, human, and rabbit enzymes.27,29,30 ACE is a
single polypeptide chain composed of two homologous
domains, each with a zinc-binding and catalytic site. As
in other species, the amino half of the bovine protein is
similar in amino acid sequence to the carboxyl half In
Figure 3 we compare the amino acid and cDNA sequence homology of bovine ACE with other published
ACE sequences. As anticipated, bovine ACE exhibits
high evolutionary conservation of both the amino acid
and the cDNA sequences.
To investigate interspecies sequence conservation
within the described ACE molecules, mouse, human,
rabbit, and bovine ACE amino acid sequences were
aligned. A consensus sequence was generated with a
unique character representing positions at which the
identical amino acid was present in each of the four
ACE sequences. A computer algorithm was then used
to center a window of 40 amino acids at each position of
the consensus sequence to quantify the number of
positions that are absolutely conserved. This analysis
(Figure 4) shows the degree of sequence identity among
the ACE proteins as a function of position along the
consensus. The positions of the two zinc-binding sites
are indicated by arrows. The results of this comparison
clearly demonstrate the evolutionary conservation
within the two domains of the ACE molecule. The
amino terminus and the carboxyl terminal domains
appear equally conserved in sequence. The largest
variation in sequence homology occurred at positions
105-143 because of a small region of the rabbit ACE
sequence that was totally nonhomologous.
Using the bovine ACE cDNA, we have undertaken a
detailed analysis of the regulated expression of ACE in
cultured bovine aortic endothelial cells. In vivo, endothelial cells exist in a confluent nonproliferating monolayer that expresses high levels of ACE. However, upon
placement in tissue culture, the cells begin to proliferate, and ACE expression is lost.10"11 It has been previously shown that growth of cultured endothelial cells to
a postconfluent state causes reappearance of ACE
activity. In the following experiments we have studied
the molecular basis for ACE expression under these
conditions: Bovine aortic endothelial cells were plated
at a subconfluent density (lx 106 cells per dish) and
grown continuously in the presence of 10% fetal calf
serum. Within 2-3 days, the cells reached confluence
and then remained viable but nonproliferative (Figure
5). The change in growth state was documented by
measurement of ['H]thymidine incorporation. As shown
in Figure 6, there was a progressive decrease in ['H]thymidine incorporation that reached a nadir at 6 days
after confluence of approximately 10% that at 2 days
before confluence (day -2). A parallel analysis of ACE
expression demonstrated an inverse relation between
[3H]thymidine incorporation and ACE activity (Figure
6). Growth-arrested endothelial cells (6 days after confluence) had approximately 12 times (range, 10-15
times; n =3) more ACE activity than actively proliferat-
Circulation Research Vol 70, No 6 June 1992
1278
Q
FIGURE 5. Photomicrographs showing endothelial cell morphology during
density-dependent growth arrest. Panel
A: At 2 days before confluence (day
-2), subconfluent cells exhibited numerous mitotic figures and cellular ex-
cA
tension. Panel B: At day 0, cells are
confluent. At day +2 (panel C) and
+4 (panel D), cells have assumed a
cobblestone appearance and remain
morphologically unchanged until the
end of the experiment.
Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017
ing cells (2 days before confluence). This increase in
ACE was not due to a generalized increase in protein
content, which did not change significantly during
growth arrest (data not shown).
We next measured the levels of ACE mRNA during
density-dependent growth arrest. ACE mRNA levels
were measured using RNase protection of antisense
bovine ACE mRNA. To control for experimental vari1
5
14;
4
9
1 3.
c
12
1 1'
0
1 0*
9,
8
Lu
0
7,-
cc
6
:
5,
CL
0
L.
r.
4,.
3,2
1 1
Days Post Confluent
FIGURE 6. Graph showing endothelial cell DNA synthesis
and angiotensin converting enzyme (ACE) activity. Bovine
aortic endothelial cells were plated at a subconfluent density
(lx]06 cells per 10-cm dish) and grown continuously for
several days. The day that cells reached confluence was
designated as day 0. ACE activity and [t'HJthymidine incorporation were measured as described in "Materials and
Methods.
"
Solid
triangles represent
the relative
?Hlthymidine
incorporation; solid squares indicate the ACE enzyme levels
expressed as units per milligram protein. Results are typical of
two experiments.
ations in the protocol, a second antisense probe, bovine
/3-actin, was included in each protection.25 As shown in
Figure 7, the levels of ACE mRNA in preconfluent cells
were very, very low. These levels increased by 21.5-fold
in 6-day postconfluent cells. Thus, there were comparable increases in ACE mRNA and ACE enzyme
activity during density-dependent growth arrest (Figures 6 and 7).
To demonstrate regulation of ACE protein expression by density-dependent growth arrest, ACE immunohistochemistry was performed on bovine endothelial
cells that were growth-arrested and then allowed to
proliferate. To perform this experiment, bovine endothelial cells were plated at high density (5 x 10i cells per
dish) in 35-mm dishes maintained at a 300 angle. This
caused immediate formation of a confluent monolayer
in the lower two thirds of the dish. After 3 days of
density-dependent growth arrest, dishes were placed
flat in the incubator. Cells at the edge of the confluent
monolayer migrated to the newly exposed area of the
dish and proliferated. As shown in Figure 8D, there was
a gradient of ACE expression in this region, with the
migrating and proliferating endothelial cells expressing
significantly less ACE than the confluent growth-arrested cells. Preimmune serum caused no significant
immunohistochemical reaction (Figure 8A). We also
observed that in the regions of densest cell growth,
endothelial cell "sprouting" had occurred (Figures 8B
and 8C). The sprouting endothelial cells formed a
reticular pattern on the surface of the monolayer. They
were readily identified by phase-contrast microscopy
and showed striking levels of ACE by immunohistochemistry. To prove that these were endothelial cells,
immunohistochemistry was performed with anti-factor
VIII antibody. The sprouting endothelial cells stained
as positively as the rest of the monolayer (data not
shown).
Shai et a! Bovine ACE cDNA Cloning and Expression
20
a)
Ar_
)
10
CD
-2
ACE
4
0
2
Days Post Confluent
mnRNA
1.0
Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017
f-Actin mRNA
2.4
7.2
1i 3.5
6
_
21..5
*
FIGURE 7. Graph and RNase assay showing that postconfluent bovine aortic endothelial cells express increased angiotensin converting enzyme (ACE) mRNA. Bovine aortic endothelial cells were plated at a subconfluent density (1 x 106 cells
per 10-cm dish) and grown continuously for several days. The
day that cells reached confluence was designated as day 0.
ACE activity was measured as described in "Materials and
Methods" and is indicated with an open triangle. Parallel
dishes of cells were used to prepare total RNA at 2 days before
confluence (day -2) and at 0, 2 4, and 6 days after rell
confluence. An RNase protection assay was used to determine
the levels of ACE mRNA. The protected ACE bands are
positioned in the figure underneath the appropriate day of cell
harvest. The protected ACE bands were quantitated by densitometry, and day -2 was assigned a value of 1.0. Relative
levels of ACE mRNA are indicated both under the protected
bands and by solid squares. These data clearly show that the
amount of ACE mRNA increases after cell confluence atnd
that this increase approximately parallels the increase in ACE
activity. Bovine fB-actin served as a control for RNase
protection and did not vary with the state of confluence of the
cells.
Discussion
The major finding of this study is that ACE mRNA is
regulated in endothelial cells in an inverse relation to
their growth state. Regulation of ACE activity in endothelium is important because of the mounting evidence
that the renin-angiotensin system is important not only
in vascular tone but also in vascular growth.31 In particular, recent findings that ACE inhibitors prevent intimal
proliferation in rat carotid artery models of balloon
injury have suggested an important role for ACE in the
pathogenesis of restenosis.32 Future experiments are
planned to document the time course for endothelial
cell ACE expression after injury.
The present study also shows that ACE has been
highly conserved during evolution. In particular, our
analysis of bovine, murine, human, and rabbit ACE
1 279
eDNA suggests that the amino and carboxyl domains of
ACE have been equally conserved. Whereas it was once
thought that ACE contained one molar equivalent of
zinc, it is now recognized that each of the two domains
of the ACE polypeptide can bind zinc and function
catalytically.3335 As demonstrated by Wei et al,35 the
two domains differ in the K,at for hydrolysis of angiotensin I: the carboxyl domain has a threefold to ninefold
higher K,3, depending on the substrate. The fact that the
testis isozyme of ACE contains only the carboxyl domain has led people to question whether the amino
domain functions in vivo. The present study supports
the idea that the amino domain has a catalytic function,
because it seems to be as evolutionarily conserved as the
carboxyl domain. These data are consistent with the
results of in vitro expression of the individual ACE
domains.35
Many recent studies have emphasized the role of the
endothelium in the homeostatic regulation of blood
pressure. One important aspect of this regulation is that
endothelium is a major source of ACE and thus acts to
produce angiotensin II. This peptide has many physiological effects including vasoconstriction, aldosterone
production by the adrenal gland, and water reabsorption by the kidney. Thus, endothelial ACE activity
should have a direct bearing on the regulation of blood
pressure.
ACE activity has been documented to be regulated by
a wide variety of stimuli in vivo and in vitro. For
example. steroids upregulate expression, whereas insulin has been reported to downregulate expression.5 One
of the most intriguing observations has been the apparent lack of endothelial cell ACE activity during cell
proliferation and increased activity during density-dependent growth arrest.'1 The fact that endothelial cells
are normally present in a nonproliferating confluent
monolayer suggests that ACE expression is a feature of
a more differentiated (i.e., functionally equivalent to in
vivo) endothelial cell phenotype. In the present study
we now show that this regulation is mediated by an
increase in steady-state mRNA levels for ACE. This was
a specific effect in that there was no change for the
constitutively expressed /3-actin mRNA.
Although the mechanisms responsible for induction
of ACE mRNA by density-dependent growth arrest
remain unknown, the immunohistochemical studies performed in this work offer some intriguing insights into a
possible role for ACE. In these studies, we demonstrated a marked increase in ACE expression in areas
where cells were growth-arrested (as predicted from our
mRNA and protein activity data) compared with proliferating areas. Of interest, in areas of very confluent
cells, the greatest amount was found in cells that
appeared to be sprouting. ACE is known to catalyze the
hydrolysis of several biologically active polypeptides in
addition to angiotensin 1.36 It is plausible that expression of a peptidase such as ACE during endothelial cell
sprouting may reflect an in vivo role in processes such as
angiogenesis.
The mechanisms responsible for increased ACE
mRNA remain unknown. Endothelial cell ACE mRNA
regulation should be an excellent model system to study
the roles of cell-to-cell contact, cell shape, cell matrix,
and cessation of cell growth in regulation of gene
expression.
1280
Circulation Research
Vol 70, No 6 June 1992
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FIGURE 8. Photomicrographs showing angiotensin converting enzyme protein expressiont in growth-arrested and proliferating
endothelial cells. Panel A: Postconfluent cells treated with rabbit preimmuine sera. There is minimal backgrouind staining. Panel
B: Postconfluent cells treated with a 1:200 dilution of a polyclonal rabbit anti-angiotensin converting enzyme antibody. There is
intense staininig of sprouting endothelial cells in an area of the plate that is 6 days postconfluenit. Panel C: High power of the
sproutinig cells. The majority of angiotensin converting enzyme activity appears to be withint these cells, althouigh the nonsprouting
cells also stain for angiotensin converting enzyme. Panel D: The tranisition zone oni the plate whlere the cells are 6 days
postconfluent (left) a nd proliferating (right). Although there is on ly a minimal increase in cell density on thle left, there is a great
increase in angiotensitn converting enzyme activity.
Acknowledgments
We wish to thank Drs. Adrian M. Tiinmers and David R.
Morris for providing the bovine /1-actin cDNA. Drs. R.S.
Kumar and G.C. Sen kindly provided us with unpublished
portions of the rabbit ACE amino acid sequence. We would
also like to thank Kimberly Langford, Kristen Frenzel, and
Yudong Zhou for their assistance.
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Bovine angiotensin converting enzyme cDNA cloning and regulation. Increased expression
during endothelial cell growth arrest.
S Y Shai, R S Fishel, B M Martin, B C Berk and K E Bernstein
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Circ Res. 1992;70:1274-1281
doi: 10.1161/01.RES.70.6.1274
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