(Received for publication, October 4, 1996, and in revised form, April 16, 1997)
From the Departments of When hypertrophic growth is induced in neonatal
rat cardiocytes by stretching, the cardiocytes express high levels of
brain-type natriuretic peptide (BNP) and the proprotein-processing
enzyme furin. A BNP precursor, When cardiocytes undergo the stretch force, they adapt by
developing cellular hypertrophy. Because cardiocytes are terminally differentiated and cannot proliferate by cell division, they exhibit hypertrophic growth, an increase in cell size and protein content. The
hypertrophic growth of cardiocytes is attained through a series of
multiple gene expressions (1-4) and phosphorylation reactions (4, 5)
as follows: expression of immediate early genes such as
c-fos, c-jun, and Egr-1; consequent
protein kinase cascade of phosphorylation reactions and expression of
peptide growth factors such as fibroblast growth factor and
transforming growth factor- Furin belongs to the yeast Kex2 endoprotease family, to which the
neuroendocrine cell-specific endoproteases PC2 and PC3 also belong (13,
14). Furin is localized on the trans-Golgi networks of virtually all
cell types including neuroendocrine cells (15). Cleavage by furin is
specific for a unique amino acid sequence, -Arg One of the furin-cleavable peptides, BNP, regulates blood pressure,
diuresis, natriuresis, and dilation of vascular smooth muscles (19,
20). In congestive heart failure and hypertrophic cardiomyopathy, BNP
is known to be produced extensively from both the atrium and ventricle.
Another important cardiac regulator, ANP, also increases these
pathological conditions in the atrium and, in severe cases, in the
ventricle where ANP is not normally expressed (21). Because the rise of
BNP is more marked than that of ANP in congestive heart failure and
hypertrophic cardiomyopathy, BNP is thought to be a more valuable
marker for those disorders than ANP (22, 23). In the atrial cardiocytes
BNP is stored together with ANP in the secretory granules, whereas
ventricular cardiocytes do not possess secretory granules, and
therefore secrete BNP, without retention in the cytoplasm, into the
bloodstream through a constitutive secretory pathway. In this pathway,
the BNP precursor is thought to be cleaved by furin, because the BNP precursor possesses a furin-cleavable RXXR sequence at its
processing site (19).
In this paper we demonstrate that both BNP and furin are highly
expressed in stretched-induced hypertrophic cardiocytes, and furin is a
major processing enzyme for the BNP precursor in rat cardiocytes.
Moreover, furin appears to control the hypertrophic growth of
cardiocytes. The prevention of hypertrophic growth by furin-specific
inhibitors was demonstrated using two protease inhibition systems,
synthetic peptidyl chloromethyl ketone (CMK) and vaccinia
vector-integrated native or variant The primary culture of neonatal ventricular
cardiocytes was prepared according to the method previously described
(24, 25), with a minor modification. Briefly, a ventricle was removed
from 1- to 3-day-old Wistar rats, rinsed with phosphate-buffered saline (PBS), minced and dispersed with 0.08% trypsin in PBS for 10 min at
37 °C, and pipetted approximately 30 times. The suspension was
lightly centrifuged, and the supernatant was removed. Three to four
further digestions with the trypsin solution were performed, and the
supernatants from each digestion were combined with Dulbecco's modified Eagle's medium (DMEM)/Ham's F12 medium (1:1, v/v, Life Technologies, Inc.) supplemented with 10% fetal bovine serum (FBS) and
50 µg/ml ampicillin. The solutions were then centrifuged at 1300 rpm
for 3 min. The pellet was gently resuspended in DMEM/F12/FBS, and the
cells were plated onto a plastic dish (100 mm, Falcon, Oxnard, CA) and
incubated at 37 °C for 90 min. During the incubation, most
non-cardiocytes attached to the floor of the dish, while cardiocytes
remained suspended. The non-attached cells were then seeded into
six-well plates with a silicon membrane bottom (FLEX I culture plates;
Flexcell Corp., McKeesport, PA) in 2 ml of DMEM/F12/FBS. The cells were
incubated for 48 h at 37 °C under a humidified atmosphere of
95% air, 5% CO2. When the cells exhibited synchronous contractions, the cells were then utilized for stretch experiments.
The silicon membrane
bottoms of the FLEX I culture plates were attached to suction tubes.
The attachment was sealed tightly with plastic glue (Taiyo Electric
Inc., Tokyo, Japan) for vacuum suction. Membrane bottoms were distended
using suction at 120 to 180 mmHg. With 120 and 180 mmHg suction, the
cells were distended approximately 10% (120 mmHg) and 15% (180 mmHg)
larger than the original size. During the experiment, suction was kept
within the range of 120-180 mmHg by periodically checking a pressure gauge.
The hypertrophic growth of
cardiocyte was assessed with the enlargement of cell size by a cell
sorter, the increase of protein synthetic rate by the incorporation of
radioisotope-labeled phenylalanine (Phe) (26, 27), and the increase of
protein/DNA ratio (27).
For cell sorter analysis, the cardiocytes were cultured for 48 h
under stretch by applying suction to the FLEX I silicon membrane plate
and then harvested with the treatment of 0.05% trypsin and 0.02%
EDTA. The cells were suspended in PBS and fixed with 2% paraformaldehyde for 30 min at 4 °C. The suspension of fixed cells was analyzed with a cell sorter (CYTORON, Ortho Diagnostic
Systems, Tokyo). A relative cell size is reflected to forward light
scatter on the x axis of a histogram (28, 29).
Protein synthesis was evaluated by three methods. First, the synthesis
was measured by the incorporation of
L-[14C]phenylalanine
([14C]-Phe) (450 mCi/mmol; NEN Life Science Products)
into a trichloroacetic acid-insoluble fraction as described previously
(24, 25). The cells were incubated for 2 and 8 h in the serum-free
DMEM/F-12. During the last 60 min incubation 1 µCi/ml
[14C]Phe was added to the incubation medium, and then the
cells were harvested after washing with PBS. The cells were dissolved
in ice-cold 20% trichloroacetic acid and precipitated by
centrifugation. The radioactivity in a trichloroacetic acid-insoluble
fraction was measured by a liquid scintillation counter.
Second, the rate of protein synthesis was measured using the combined
equilibrium and pulse labeling method (26, 27). For equilibrium
labeling the cardiocytes were cultured with or without stretch for 4 days in the DMEM/F-12 medium with 10% FBS containing 0.1 µCi/ml
[14C]Phe (450 mCi/mmol; NEN Life Science Products).
Equilibrium labeling is reported to require 4 to 5 days' culture of
cardiocytes to attain a constant specific radioactivity (30). The
medium containing [14C]Phe was changed 4 times every
24 h during equilibrium labeling. After the equilibrium labeling,
pulse labeling was performed by incubating the cells with or without
stretch for 4 h in the same medium containing another
radioisotope, 2.5 µCi/ml [3H]Phe (40-60 Ci/mmol; NEN
Life Science Products). The cells were harvested after washing with PBS
and dissolved in ice-cold 20% trichloroacetic acid and precipitated by
centrifugation. Precipitated protein was hydrolyzed with 6 N HCl at 110 °C for 24 h. Phe in the protein
hydrolysate and in the labeling medium was separated by high pressure
liquid chromatography (HPLC). Separated samples were post-labeled with
O-phthalaldehyde for fluorodetection. The specific
radioactivity of Phe was determined by a liquid scintillation counter.
The rate of protein synthesis (Ks) was calculated by
Equation 1.
Molecular Medicine and
Cell Biology,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
BNP, possesses a furin-cleavable
Arg-X-X-Arg motif, which is cleaved when
BNP is
processed to form BNP-45. The Arg-X-X-Arg motif is found in
many precursors of growth factors and growth-related proteins. To
determine if furin converts
BNP to BNP-45 as well as other
unidentified growth-promoting protein precursors to their active form
that may induce hypertrophic growth in cardiocytes, we used two
protease inhibitor systems, synthetic peptidyl chloromethyl ketones
(CMK) (dec-Arg-Val-Lys-Arg-CMK and dec-Phe-Ala-Lys-Arg-CMK; where dec
is decanoyl) and vaccinia vector-integrated native and variant
1-antitrypsins. The furin-specific inhibitors, dec-Arg-Val-Lys-Arg-CMK and variant
1-antitrypsin with
the inhibitory determinant Arg-X-X-Arg, suppressed the
stretch-induced hypertrophic growth of cardiocytes as well as the
processing of
BNP to BNP-45. The other serine protease inhibitors
and variant
1-antitrypsin against elastase, or thrombin,
however, neither suppressed the hypertrophic growth nor prevented the
processing of
BNP to BNP-45. Thus, we suggest that furin catalyzes
the conversion of
BNP to BNP-45 as well as growth-promoting
proproteins to their active form, which might induce hypertrophic
growth in cardiocytes.
(TGF-
)1; cardiac
regulatory peptides such as atrial natriuretic peptide (ANP) and
brain-type natriuretic peptide (BNP); the contractile proteins myosin
light chain-2 and fetal-type
-myosin heavy chain; and probably
extracellular matrix-degrading metalloproteinases at a remodeling step
(6). Some these proteins are produced as precursors, including TGF-
(7, 8), ANP (9, 10), BNP (10), and metalloproteinases including
membrane-type matrix metalloproteinase (11) and stromelysin-3 (12). The
precursors are cleaved to the active forms by endoproteases. Because
TGF-
, BNP, membrane-type matrix metalloproteinase, and stromelysin-3 contain a cleavage site for the proprotein-processing endoprotease furin (7, 10-12), we examined whether furin might be another candidate
gene expressed during the hypertrophic growth of cardiocytes.
4-X-X-Arg
1
X+2 (16). An additional basic amino acid at the
2 or
6
position facilitates more efficient cleavage (17). This furin-specific Arg-X-X-Arg (RXXR) motif is found in many
growth-promoting peptides or proteins as listed above. In addition to
this cleavage reaction, furin is known to exhibit another function,
recycling between the trans-Golgi network and plasma membrane (15, 18).
With this recycling, furin expression may increase protein flow through a constitutive pathway, which results in an augmented transport of
growth-promoting peptides or proteins outside the cells.
1-antitrypsins.
Cell Culture
where P* is the amino acid specific radioactivity in
the protein, and F* is the specific radioactivity in the
precursor. t1 is the time when
[3H]Phe is added, in this experiment
t1 = 0. t2 is the
duration time when protein was pulse-labeled with
[3H]Phe, in this experiment t2 = 4 h.
(Eq. 1)
Third, the protein/DNA ratio of cardiocytes was obtained from the
values of protein and DNA per dish (5 × 105 cells).
The total DNA content in homogenated cells was determined using a
fluorescent dye 4,6-diamidino-2-phenylindole (31). 4
,6-Diamidino-2-phenylindole was dissolved at 100 ng/ml in the buffer
100 mM NaCl, 10 mM EDTA, and 10 mM
Tris, pH 7.0. Salmon sperm DNA was used as a standard. Fluorescence was
measured by excitation at 360 nm and emission at 450 nm. Protein
concentration was measured using the Bradford method (Bio-Rad) using
bovine serum albumin as a standard.
Radioimmunoassay (RIA) was carried out using an RIA kit for rat BNP-45 (RIK 9085, Peninsula Laboratories, Belmont, CA), which utilizes an antibody to rat BNP-45 generated in rabbits in response to a synthetic rat BNP-45 (32, 33). The antibody was added to test samples and incubated for 16-24 h at 4 °C. 125I-Labeled BNP (between 1 × 104 and 1.5 × 104 cpm) was then added, mixed, and incubated again at 4 °C for 16-24 h. The second antibody (goat anti-rabbit immunoglobulin G serum mixed with normal goat serum) was added to the above mixture and then incubated at room temperature for 90 min. RIA buffer supplied with the kit was added, and the samples were vortexed and centrifuged for 20 min at 3000 rpm. The supernatant was removed, and the radioactivity in the pellets was counted in a gamma counter.
BNP Extraction and HPLC AnalysisBNP was extracted from cardiocytes or culture medium. For the BNP extraction from cardiocytes, the cells were scraped from the silicon membrane plates and homogenized in 5 ml of ice-cold 5 N acetic acid (containing 0.3 mg/ml phenylmethylsulfonyl fluoride) using a ground-glass homogenizer, as described previously (32). The supernatants, obtained after centrifugation at 100,000 × g for 30 min, were loaded onto a Sep-Pak C18 cartridge (Millipore, Milfold, MA). For the extraction from the culture medium the medium was lightly centrifuged to remove contaminating cells or cell debris, then lyophilized, and reconstituted to 4 ml by adding 4% acetic acid. The acid solution was passed through a Sep-Pak C18 cartridge.
After washing the cartridge with 0.5 M acetic acid,
adsorbed peptides were eluted with 60% acetonitrile containing 0.1%
trifluoroacetic acid. Each eluate was evaporated under vacuum prior to
lyophilization. Lyophilized samples were dissolved in 0.1%
trifluoroacetic acid and applied to a reverse-phase HPLC analyzer
(Hitachi L-6200). HPLC analysis was carried out using a nucleosil 10C18
column (4.6 × 150 mm, Chemco Pak, Osaka, Japan). The column was
equilibrated with 0.1% trifluoroacetic acid and eluted with a linear
gradient of 0-60% acetonitrile in 0.1% trifluoroacetic acid at a
flow rate of 1 ml/min. Aliquots of all fractions were submitted to RIA
for BNP. The endogenous converting activity of BNP to BNP-45 in
cardiocytes was assessed by measuring the
BNP and BNP-45 fractions
in cardiocyte lysates and those released into the culture medium from
the cardiocytes. The ratio between the two fractions was determined by
separating the immunoreactive (ir) BNP on HPLC (34).
Total RNA was isolated from cardiocytes and electrophoresed on a 1% agarose gel as described previously (34, 35). RNA electrophoretic bands were blotted onto a nylon membrane (Hybond-N, Amersham Japan, Tokyo, Japan). Hybridization was performed in a solution consisting of 5 × SSPE (20 × SSPE, 3.6 M NaCl, 200 mM NaH2PO4, 20 mM EDTA, pH 7.4), 10 × Denhardt's reagent (0.2% Ficoll, 0.2% polyvinylpyrrolidone, 0.2% bovine serum albumin), 50% formamide, 1.4% sodium dodecyl sulfate (SDS), and 0.1 mg/ml salmon sperm DNA with either a 32P-labeled mouse furin cDNA fragment (924 base pairs; 36), or the 628-base pair rat BNP cDNA fragment (37). After washing, the membrane was exposed to x-ray film (Kodak, XAR 5).
Western Blot AnalysisFor immunodetection of furin, the cells were cultured and harvested for preparation of cell lysates in lysis buffer (70 mM Tris-HCl, pH 6.8, 11.2% glycerol, 3% SDS, 0.01% bromphenol blue, 5% 2-mercaptoethanol). Cell lysates were electrophoresed on a 7.5% polyacrylamide gel under the reducing condition. Separated proteins were blotted onto a nitrocellulose membrane and probed with rabbit anti-furin antiserum (ST-73) at 1:5000 dilution. Furin blots were detected using an enhanced chemiluminescence detection system (Amersham, Buckinghamshire, UK).
Immunocytochemical StudiesImmunostaining of furin was performed as described previously (34, 35). Briefly, the cells were cultured on the silicon membrane plate. After half of the membrane bottoms were stretched, stretched and non-stretched cells were fixed with 2% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for 1 h at room temperature. The specimens were incubated for 2 h with rabbit anti-furin serum (ST-73) and then incubated with indodicarbocyanine-conjugated affinity-purified donkey anti-rabbit immunoglobulin G (Jackson Immuno Research Lab, West Grove, PA), diluted with the blocking buffer (1% bovine serum albumin, 0.2% skim milk, 0.5% Triton X-100, 0.1% sodium azide), and incubated for 2 h, followed by mounting. The specimens were examined with an Olympus BX50 microscope with an incident illuminator.
Enzyme AssayProcessing enzyme activities in cardiocytes was assessed by using the three enzyme substrates pyroglutamyl-arginylthreonyl-lysyl-arginyl-methylcoumarylamide (pyr-Arg-Thr-Lys-Arg-MCA), t-butyloxycarbonyl-alanyl-glycyl-prolyl-argininyl-methylcoumarylamide (boc-Ala-Gly-Pro-Arg-MCA), and t-butyloxycarbonyl-glycyl-lysyl-argininyl-methylcoumarylamide (boc-Gly-Lys-Arg-MCA) (Peptide Institute, Inc., Osaka, Japan). The substrates pyr-Arg-Thr-Lys-Arg-MCA, boc-Ala-Gly-Pro-Arg-MCA, and boc-Gly-Lys-Arg-MCA are designed to assess furin, rat ANP precursor processing enzyme, and paired basic residue cleavage enzyme, respectively. The enzyme activity was measured by the production of liberated fluorescent aminomethylcoumarin (AMC) from the carboxyl terminus of a synthetic peptide substrate (34, 38). The enzyme source was prepared by sonicating cultured cardiocytes in PBS containing 1 mM CaCl2 and 2% n-octylglucoside, as described previously by Garten et al. (39). To 0.20 ml of cell lysate 20 nmol of a synthetic substrate was added to make a total volume of 0.25 ml consisting of 50 mM HEPES, pH 7.3, 1 mM CaCl2, and 2% n-octylglucoside. The enzyme reaction was performed at 37 °C for 1 h and then 2.0 ml of 5 mM EDTA was added to stop the reaction. The liberated AMC was fluorometrically measured by excitation at 380 nm and emission at 460 nm.
Effect of Protease Inhibitors on Furin-type EnzymeThe potency of other processing enzyme inhibitors decanoyl-arginyl-valyl-lysyl-arginyl-chloromethyl ketone (dec-Arg-Val-Lys-Arg-CMK) and decanoyl-phenyl-alanyl-lysyl-arginyl-chloromethyl ketone (dec-Phe-Ala-Lys-Arg-CMK) (a kind gift of Dr. W. Garten, Marburg) was assessed by adding these inhibitors (up to 50 µM) to the above described enzyme assay of cardiocyte lysates (39, 40). The inhibitor dec-Arg-Val-Lys-Arg-CMK inhibits furin; and dec-Phe-Ala-Lys-Arg-CMK inhibits paired basic residue cleavage enzyme.
Another inhibition system of furin cleavage is the vaccinia virus
recombinants integrating native and variant
1-antitrypsins (41, 42). Native
1-antitrypsin and other two variant
1-antitrypsins contain serpin (serin
protease inhibitor)-specific determinant sequences Ala-Ile-Pro-Met, Ala-Ile-Pro-Arg, and Arg-Ile-Pro-Arg, and
each sequence inhibits elastase, thrombin, or furin, respectively. Vaccinia virus (VV) recombinants were used: VV:
1-NAT
(normal
1-antitrypsin), VV:
1-PIT (natural
variant of
1-antitrypsin-Pittsburgh containing
Ala-Ile-Pro-Arg determinant), and VV:
1-PDX (engineered mutant of
1-antitrypsin-Pittsburgh containing
Arg-Ile-Pro-Arg determinant) (41, 42). The cardiocytes were incubated
with the wild type VV strain (WR; ATCC VR-119) and three other vaccinia recombinants for 1 h (m.o.i. = 5, 20, or 50), and then the cells were washed with PBS-M (PBS with 1 mM MgCl2) 3 times for subsequent culture. After culturing the cells under
stretching for 4 h the cells were harvested for the processing
enzyme assay.
To examine the effect of
the peptidyl-CMK on the stretched-induced hypertrophic growth of
cardiocytes, the cells were incubated with the inhibitor for 4 h
at a concentration of 50 µM. To examine the effect of
1-antitrypsins on hypertrophic growth, the cells were
incubated with wild strain WR, VV:
1-NAT,
VV:
1-PIT, or VV:
1-PDX for 1 h
(m.o.i. = 5, 20, or 50). The viral solution was washed out with PBS-M;
DMEM/F-12/FBS was added; the cells were kept stretching for another
4 h; and the cells were harvested and applied to the cell sorter
for analysis.
To examine the inhibitory effect of the peptidyl-CMKs on the conversion
of BNP to BNP-45, cardiocytes were cultured for 8 h in the
presence or absence of each peptidyl-CMK at a final concentration of 25 or 50 µM. Peptidyl-CMK was added 2 times at a 4-h
interval because the half-life of the inhibitor is thought to be 4-8 h (39, 40). The effects of the trypsin inhibitors on the conversion of
BNP to BNP-45 were also examined. The cells were cultured for 4 h in the presence or absence of
L-1-chloro-3-(4-tosylamido)-7-amino-2-heptanone (TLCK) and
fungal serin protease inhibitor leupeptin. Each inhibitor was added to
the DMEM/F-12 culture medium at a final concentration of 100 µM. When the cells had been cultured for 4 h the
culture medium was collected for HPLC analysis. The effect of
1-antitrypsins on the BNP processing was performed as
described above. After stretching the medium was collected for HPLC
analysis.
We evaluated the
stretch-induced hypertrophic growth of cardiocytes by four different
experiments. The first method, measurement of cell size by a cell
sorter has been utilized for evaluating hypertrophy of vascular smooth
muscle cells and renal epithelial cells (28, 29, 43, 44). Cardiocytes
were plated almost to confluency on a silicon membrane plate. The cells
exhibited synchronous contractions at approximately 100-120 beats/min.
Stretching of cardiocytes has been used for their hypertrophic growth
(5, 24, 25, 27). The cells were stretched 10-15% longer than their
original size for 48 h by pulling the membrane bottom with suction. The cells, with or without stretching, were harvested and
applied to the cell sorter analysis to obtain a histogram of their
size. When removed from the plate, spindle-shaped cardiocytes on a
silicon membrane became rounded by microscopic observation (Fig.
1A). Approximately 4 × 103 cells were analyzed to obtain each histogram. The cell
size is reflected to forward light scatter on the x axis of
a histogram as described previously (28, 29). The histogram of
stretched and non-stretched cells exhibited a bell-shaped distribution
and that of stretched cells shifted to the right from that of
non-stretched cells (Fig. 1B). The forward light scatter
before stretching was 131.1 ± 26.8, whereas that after stretching
was 141.6 ± 26.3. The difference was significant
(p < 0.001) between the stretched and non-stretched
cells as assessed by the two sample t test.
Second, hypertrophic growth of cardiocytes was measured by the
incorporation of [14C]Phe into cellular protein (24, 25).
The incorporation of [14C]Phe was approximately 2-fold in
stretched cells than in non-stretched cells after only 2 h of
stretching (Fig. 2A,
p < 0.01). This increase continued even 8 h after
the start of stretching. Third, we compared the rate of protein
synthesis between stretched and non-stretched cardiocytes by using the
equilibrium and pulse labeling method (26, 27). The rate was calculated
by Equation 1 under "Experimental Procedures" from the ratio of the
pulse labeling with [3H]Phe and equilibrium labeling with
[14C]Phe in cellular protein and free
radioisotope-labeled Phe in the culture medium (Table
I). The protein synthetic rate
Ks of stretched cardiocytes (0.0305 ± 0.0009)
was clearly larger than that of non-stretched cardiocytes (0.0266 ± 0.0020). Total cellular protein was increased approximately 20%
more than that in non-stretched cardiocytes during 4-day stretching
(Table I). Fourth, the protein/DNA ratio of cardiocytes was evaluated
by 4-h stretching. The ratio for stretched cardiocytes was
approximately 20% greater than that of non-stretched cardiocytes (Fig.
2B). Thus, stretching significantly increased the cell size
and protein synthesis in cultured cardiocytes as described previously
(24, 25, 27).
|
Total RNA was isolated from
cardiocytes after stretching for 48 h and then applied to Northern
blot analysis. Fig. 3A shows that BNP mRNA was slightly visible in non-stretched cells, whereas the mRNA became more intense in stretched cardiocytes. In contrast, -actin mRNA stayed in a similar level between stretched and
non-stretched cardiocytes. Consistent with the BNP mRNA expression,
the stretching induced approximately a 2-fold increase in the release
of immunoreactive BNP (irBNP) into the culture medium (Fig.
3B). The rat BNP-45 corresponds to the position 51-90 amino
acid region of the 90-residue
BNP (37). The molecular form of BNP
was not markedly affected by stretching. In the culture medium of both
stretched and non-stretched cardiocytes only a processed form of BNP-45
was observed. irBNP in the cell extracts from stretched and
non-stretched cardiocytes consisted of two molecular forms, a major
form of a processed BNP-45 and a minor form of a precursor
BNP (Fig.
3C). Although there was no remarkable difference in
molecular forms of irBNP between stretched and non-stretched
cardiocytes, the proportion of BNP-45 to
BNP appeared a little
higher in the cell extract of stretched cardiocytes.
Stretch-induced Expression of Furin
We then examined the
effect of stretching on the expression of furin. There were some weakly
furin-stained cells in non-stretched cardiocytes (Fig.
4A), whereas there were many
extensively furin-stained cells in stretched cardiocytes (Fig.
4B). Furin was localized punctately around perinuclear
region to cytoplasmic area. Although furin localizes on the Golgi
complexes, its overexpression may extend its localization over the
cytoplasm due to its extensive recycling between the trans-Golgi
networks and plasma membrane (15, 18). The furin mRNA band was
lightly visible in non-stretched cardiocytes. Furin mRNA was
increased abundantly in 48-h stretched cardiocytes (Fig.
4C). In contrast, a -actin mRNA level was not changed
by stretching. Furthermore, the immunoblot of furin about 100 kDa in
size was stronger in stretched cardiocytes than in non-stretched
cardiocytes (Fig. 4D). These findings that both mRNA and
protein levels of furin became more abundant in stretched cells are
consistent with the increased immunocytochemical staining in Fig.
4B. Thus, both BNP and furin were co-elevated with the hypertrophic growth of cardiocytes.
Protease Activities in Cardiocytes
We assayed the proteolytic
activities of the cardiocyte lysates by using three enzyme substrates:
boc-Ala-Gly-Pro-Arg-MCA, which is a substrate for rat ANP precursor
processing enzyme; boc-Gly-Lys-Arg-MCA, which is a substrate for paired
basic residue cleavage enzyme; and pyr-Arg-Thr-Lys-Arg-MCA, which is
designed to serve as a substrate for furin. When the proteolytic
activity observed on the ANP precursor-processing enzyme substrate
boc-Ala-Gly-Pro-Arg-MCA was set to equal 100%, the activity for the
dibasic residue cleavage enzyme substrate was approximately 95%, while
the activity for furin substrate was approximately 430% (Fig.
5A). Thus, cardiocyte lysates
contained high cleavage activity to a furin-type substrate. However, we
could not attribute this cleavage activity directly to the furin by the
following two reasons. First, the cleavage activity of the furin
substrate may be overestimated because the substrate
pyr-Arg-Thr-Lys-Arg-MCA is cleaved not only by furin but also by
monobasic- or dibasic-site cleavage enzymes. Second, cardiocyte
extracts contain at least another furin type yeast Kex2 endoprotease
PACE4, although its substrate specificity is narrowly limited compared
with that of furin (34, 45, 46). Cardiocytes contain more than one form
of furin-type enzymes so that we need to purify this activity for its
accurate characterization.
Since yeast Kex2 family endoproteases including furin and PACE4 require calcium for their activity, we examined the calcium dependence of this activity in non-stretched cardiocytes. The activity was maximal at 1 mM Ca2+ and then decreased with the increment of Ca2+ concentrations up to 10 mM (data not shown). Then the calcium chelator EDTA was added to the enzyme reaction. The activity was inhibited 70% in the presence of 5 mM EDTA (Fig. 5B). The calcium-dependent activity was more elevated than calcium-independent activity in stretched cardiocytes. This finding also suggested that the furin-type activity is elevated during hypertrophic growth of cardiocytes.
When the furin-type endoprotease inhibitor dec-Arg-Val-Lys-Arg-CMK was added to the enzyme reaction mixture, the peptidyl-CMK inhibited the liberation of AMC from the substrate pyr-Arg-Thr-Lys-Arg-MCA. An inhibitor concentration of 25 µM inhibited proteolysis of the furin substrate by approximately 73%, and a concentration of 50 µM produced approximately 87% inhibition (Table II). In contrast, the dibasic site cleavage enzyme inhibitor dec-Phe-Ala-Lys-Arg-CMK inhibited proteolysis of the furin substrate by approximately 28% at 25 µM and 44% at 50 µM. Thus, cardiocyte lysates contain high proteolytic activity of furin-type enzyme.
|
Because furin was highly
expressed with the hypertrophic growth of cardiocytes, we wondered if
furin plays an essential role in inducing hypertrophic growth in
cardiocytes. To examine this possibility, we utilized two furin-type
endoprotease inhibitors, synthetic peptidyl inhibitor
dec-Arg-Val-Lys-Arg-CMK and natural 1-antitrypsin and
its variants integrated in the vaccinia viral gene transfer system.
First, we used dec-Arg-Val-Lys-Arg-CMK, which inhibits a furin-type
enzyme. When cardiocytes were cultured without stretch in the presence
or absence of dec-Arg-Val-Lys-Arg-CMK at a concentration of 50 µM, the histograms of cardiocytes by forward light
scatter had a similar distribution with the average size of 142.8 ± 25.3 in the presence of the inhibitor and 143.6 ± 26.8 in the
absence of the inhibitor (Fig. 6,
A and B). When cardiocytes were stretched in the
presence or absence of the inhibitor, the average forward light scatter
was 143.3 ± 26.9 with the inhibitor and 149.8 ± 25.2 without the inhibitor (Fig. 6, C and D). We
evaluated the difference in cell size against the non-stretched cells
cultured without the inhibitor (Fig. 6A) using the two
sample t test. There was no difference in the size of the
cells that were not stretched (p > 0.05; Fig.
6B) nor were the stretched cells that were cultured with the
inhibitor (p > 0.05; Fig. 6D). There was a
significant difference, however, between the stretched cells cultured
with and without the inhibitor (Fig. 6, C and D,
p < 0.001). Thus, even under the stretch force, the
inhibitor prevented the hypertrophic growth of cardiocytes (Fig.
6C). Unfortunately we used up another peptidyl-CMK,
dec-Phe-Ala-Lys-Arg-CMK, for the BNP processing experiment and could
not examine the effect of the inhibitor against dibasic residue
cleavage enzyme.
We then examined the effect of the peptidyl-CMKs on protein synthesis
of stretched cardiocytes by two methods. First, we measured the
incorporation of [14C]Phe in cellular protein in a
similar manner to Fig. 2A. In the presence of 50 µM peptidyl-CMKs, especially furin-specific
dec-Arg-Val-Lys-Arg-CMK, the increase of [14C]Phe
incorporation was suppressed over 50% at both 2- and 8-h points (Fig.
7A). Second, the ratio of
total protein/DNA in stretched cardiocytes was also suppressed to the
level of non-stretched cardiocytes by this furin-type enzyme inhibitor
(Fig. 7B). Thus, the inhibitory effect of furin-type enzyme
inhibitor on hypertrophic growth of cardiocytes was demonstrated by
cell sorter histogram as well as by protein synthesis.
To confirm further the suppressive effect of furin-type endoprotease
inhibitors on the hypertrophic growth, we used another type of
inhibitor, natural 1-antitrypsin and its variants.
Vaccinia virus infection results in the rapid degradation of cellular
mRNAs for the effective translation of viral mRNAs and causes
cell lysis 48-72 h following infection (47, 48). Thus, we observed the effect of
1-antitrypsin only 4 h after the
infection. An expression experiment at 4 h after vaccinia
infection is earlier than usual, compared with regular vaccinia gene
expression experiments that are usually performed at 12-24 h after
infection (48). The cardiocytes, while being stretched, were exposed to
wild strain WR or each of vaccinia recombinant
1-antitrypsins for 1 h. The cells were kept
stretching for another 4 h, and then the activity to cleave the
furin-type substrate pyr-Arg-Thr-Lys-Arg-MCA was measured in the
cardiocyte lysate. Table III shows that
the cardiocyte lysate infected with VV:
1-NAT and
VV:
1-PIT exhibited slightly less activity compared with
that infected with the WR strain. In contrast, the cardiocyte lysate
infected with VV:
1-PDX demonstrated only 9% control
activity (WR). Thus, the furin-type inhibitor
1-PDX effectively decreased the activity to cleave
pyr-Arg-Thr-Lys-Arg-MCA.
|
Subsequently, we assessed the effect of the vaccinia recombinant
1-antitrypsins on the size of stretched cardiocytes by
using cell sorter analysis (Fig. 8).
Infection of VV:
1-NAT or VV:
1-PIT did not
affect the stretch-induced enlargement of cardiocyte size compared with
the control cell size infected with the WR strain (WR strain,
133.4 ± 69.4; VV:
1-NAT, 132.2 ± 72.0;
VV:
1-PIT, 132.4 ± 70.4, by forward light scatter).
In contrast, infection of VV:
1-PDX induced the
suppression of the stretching-induced cell size enlargement (126.6 ± 66.6 by forward light scatter, p < 0.001 against
the cells infected with the WR strain). The histogram of forward light
scatter, however, was not bell-shaped but was distributed to the right
with a peak to the left asymmetrically similar to a log normal
distribution curve. Thus, the standard deviation became much larger
than that shown in Fig. 6. We examined several different virus titers
for infecting cardiocytes (m.o.i. = 5, 20, 50), but a histogram
consistently revealed an asymmetrical distribution with any virus
titer. The deformation of cell shape may be derived by the degradation
of cytoskeleton, such as actin and tubulin mRNAs (49). Although the
histogram of cell size was affected by vaccinia viral infection, the
average size of cardiocytes expressing
1-PDX stayed
smaller after stretching than that of cells expressing the WR strain,
1-NAT, and
1-PIT with the same
statistical analysis used for Figs. 1B and 6. Thus, two
different types of inhibitors against a furin-type enzyme neutralized
the effect of stretching on the hypertrophic growth of cultured
cardiocytes.
The Effect of Trypsin Inhibitors, Peptidyl-CMKs, and
We
observed co-elevation of BNP-45 and furin in the stretched cardiocytes
(Figs. 3 and 4), but these data do not directly present evidence that
furin catalyzes the conversion of BNP to BNP-45. To obtain direct
evidence, we used several endoprotease inhibitors, trypsin inhibitors
including leupeptin and TLCK, peptidyl CMKs, and vaccinia
vector-integrated
1-antitrypsins. Leupeptin and TLCK
inhibited less than 10% of the proteolytic activity to pyr-Arg-Thr-Lys-Arg-MCA in cardiocyte lysates (data not shown). These
trypsin inhibitors were added to the cardiocyte culture to examine
their effect on the processing of
BNP to BNP-45. The culture medium
of cardiocytes without the inhibitor exhibited a single peak of BNP-45
as determined by HPLC (Fig. 3C). The addition of the
inhibitors to the cardiocyte culture produced a tiny fraction of
BNP
as assessed with HPLC separation (Fig.
9), indicating that each inhibitor was
not sufficient to suppress the conversion to BNP-45. In contrast, the
addition of dec-Arg-Val-Lys-Arg-CMK (25 µM) produced some
uncleaved
BNP, and a higher concentration (50 µM)
produced much less conversion of
BNP to BNP-45 (Fig. 10). But a dibasic site cleavage enzyme
inhibitor (dec-Phe-Ala-Lys-Arg-CMK) did not affect the conversion of
BNP, even at 50 µM.
We then examined the processing of BNP in cardiocytes by expressing
three types of 1-antitrypsins in cultured cardiocytes. When the WR strain, VV:
1-NAT, and
VV:
1-PIT were infected, cardiocytes secreted only a
processed form of BNP-45 without the precursor form
BNP. In
contrast, expression of
1-PDX in the cardiocytes resulted in the appearance of
BNP (Fig.
11). The proportion of
BNP to BNP-45
was greater in
1-PDX-expressing cardiocytes than in
those cultured in the presence of dec-Arg-Val-Lys-Arg-CMK. Thus, the
two types of furin-specific inhibitors, peptidyl-CMK as well as the
variant
1-antitrypsin, prevented the conversion of
BNP to BNP-45, while other serin protease inhibitors did not.
The present results indicate that 1) both BNP and furin are highly
expressed in cardiocytes hypertrophied by stretching; 2) furin-specific
inhibitors dec-Arg-Val-Lys-Arg-CMK and variant 1-antitrypsin
1-PDX suppressed the
stretch-induced hypertrophic growth of cardiocytes, as well as the
conversion of the BNP precursor form (
BNP) to the processed form
(BNP-45).
Cardiocytes are known to exhibit hypertrophic growth in response to
stretch force and pressure overload (1, 4, 24, 25, 27). We demonstrated
stretch-dependent hypertrophic growth of cardiocytes using
two methods, cell sorter analysis and protein synthesis (Figs. 1 and 2,
Table I). The mechanical stress and some of the peptide growth factors
are known to induce the expression of the immediate early genes
c-fos, c-jun, c-myc, and
egr-1 in cardiocytes (1-4, 24, 50). Consequently, these
immediate early genes express several embryonal stage-specific genes
including myosin light chain-2, -myosin heavy chain, ANP, and
mitogen-activated protein kinases, which are thought to be responsible
for cellular hypertrophy (1-4, 25). Another natriuretic peptide BNP
mRNA is elevated as rapidly as the immediate early genes but much
faster than ANP mRNA does during the hypertrophic growth of
cardiocytes (51). BNP as well as ANP serve to reduce extracellular body fluid to compensate for the pressure overload to the myocardium by
inducing natriuresis, diuresis, and vasodilation (20). We confirmed the
elevation of BNP mRNA and irBNP in stretched cardiocytes (Fig.
3).
Along with BNP, furin was also increased with hypertrophic growth of
cardiocytes (Fig. 4). We assayed basic residue cleavage enzymes using
the three substrates. Each fluorescent MCA-coupled peptide was used as
a substrate for either the rat ANP precursor-processing enzyme, the
paired basic residue cleavage enzyme, or furin (34, 38). The
cardiocytes contained a high level of proteolytic activity that cleaves
the furin-type substrate, and this activity was enhanced by stretching
(Fig. 5). The furin-type proteolytic activity was extensively inhibited
by two furin-specific inhibitors, dec-Arg-Val-Lys-Arg-CMK, and vaccinia
virus-integrated variant 1-antitrypsin with
Arg-X-X-Arg determinant,
1-PDX. These
furin-specific inhibitors not only prevented the conversion of
BNP
to BNP-45 (Figs. 10 and 11) but also suppressed the hypertrophic growth
of cardiocytes (Figs. 6, 7, 8). Furin is a membrane protein localized in
the trans-Golgi networks and recycles between the trans-Golgi networks
and plasma membranes (15, 18), whereas
1-antitrypsin is
a secretory protein (52).
1-PDX may inhibit furin
activity at the trans-Golgi networks or during traffic to the plasma
membrane. In addition, since furin is known to be secreted after
truncation from the membrane-bound domain (53), we cannot exclude the
possibility that
1-PDX inhibits furin action outside the
cells. Further study is required to pinpoint a site where
1-PDX inhibits furin activity.
Furin exerts an essential role in cell proliferation and
de-differentiation by cleaving the RXXR motif on
growth-related precursor peptides or proteins (13, 36). The
RXXR motif is found in several cardiovascular regulatory
peptide precursors including the amino terminus of BNP and CNP (10),
and both amino and carboxyl termini of adrenomedullin (54), big
endothelin (55), and parathyroid hormone-related protein (PTHrP) (56).
This motif is also found in a variety of many other precursors for
growth-promoting peptides or proteins (57). Cardiocytes produce
regulatory peptides with the RXXR motif including BNP (19),
TGF- (58), endothelin (59), and PTHrP (60). Each peptide precursor
undergoes a variety of distinct proteolytic reactions. BNP precursor
BNP consists of 108 amino acids in humans, 105 in pigs, and 95 in
rats.
BNP is cleaved to a distinct species-specific size at the
RXXR motif; 32 amino acid BNP in humans, 26 and 32 amino
acids BNPs in pigs, and 45 amino acid BNP in rats (10). The processing
of
BNP to BNP-45 is predicted to be carried out by furin (19). We
previously demonstrated that BNP is co-elevated with furin but not with
PACE4 in the same region of rat atrial and ventricular tissue after myocardial infarction (34). In the present study, the processing of BNP
by furin was clearly proved by using two types of furin inhibitors
(Figs. 9, 10, 11). TGF-
is activated through at least two proteolytic
steps, cleavage of the precursor by furin, and removal of latent
TGF-
binding protein from a 25-kDa TGF-
dimer probably by plasmin
(7, 8). TGF-
does not appear to induce hypertrophic growth of
cardiac cells but rather maintains the contractile function of
cardiocytes (58). Recently endothelin was found to be produced from
cardiocytes (59). Endothelin requires two processing reactions,
proteolysis of the precursor to big endothelin by furin, followed by
the cleavage of big endothelin by endothelin-converting enzyme-1 (61).
The conversion of big endothelin to mature endothelin by
endothelin-converting enzyme-1 is efficient (62). The first cleavage
step of endothelin precursor to big endothelin by furin may be more
rate-limiting because furin expression in cardiocytes is regulated by
pressure load and stretching in cardiocytes (34). PTHrP is also known
to be produced from cardiocytes and processed to mature form by furin
(56, 60). PTHrP was shown to antagonize the creatine kinase-inducing
action of parathyroid hormone in cardiocytes (63). It is not known whether PTHrP exhibits hypertrophic growth in cardiocytes, although PTHrP has growth-promoting activity in some studies (64).
In addition, cardiocytes produce ANP and angiotensinogen that require another type of processing reaction (20, 65). ANP appears to be cleaved by protease that is probably membrane-associated (66, 67). Formation of angiotensin II requires three proteolytic steps for its activation, cleavage of angiotensin I from angiotensinogen by renin, proteolytic activation of prorenin to renin by renin-converting enzyme, and conversion of decapeptide angiotensin I to octapeptide angiotensin II by angiotensin I-converting enzyme (65). In patients with chronic heart failure, angiotensin I-converting enzyme message was elevated 3-fold compared with normal individuals (68). But, this enzyme was not induced in stretched cardiocytes (69), in contrast to the increase of furin-type protease in these cells. Another type of angiotensin I-converting enzyme, chymase, also exerts its action in failing hearts (70), although chymase is localized in mast cells contained in the heart (71). With these examples, cardiocytes contain many kinds of processing proteases.
Among these processing enzymes we suggest that furin might have an essential role in the control of the hypertrophic growth of cardiocytes by generating bioactive peptides or proteins, including a number of growth factors and cardiovascular regulatory peptides.
We gratefully acknowledge Dr. Wolfgang
Garten, Institut für Virologie, Phillips-Universität
Marburg, Germany, for providing us with peptidyl-CMKs; Dr. Gary Thomas,
the Vollum Institute, Oregon Health Science University, Portland, OR,
for vaccinia recombinant 1-antitrypsins; Dr. Naoto
Minamino, National Cardiovascular Center Research Institute for rat BNP
cDNA; Reiko Shizuka for assistance in operating the cell sorter
CYTORON; and Mina Takei for secretarial assistance.