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National Cardiovascular Center Research Institute (Y.I., N.M., T.K., K.K., H.M.), Fujishirodai, Suita, Osaka 565; Faculty of Pharmaceutical Sciences, Setsunan University (Y.I., M.Y.), Hirakata, Osaka 573–01; and Shionogi Co., Ltd. (T.T.), Mishima, Settsu, Osaka 566, Japan
Address all correspondence and requests for reprints to: Naoto Minamino, Ph.D., National Cardiovascular Center Research Institute, 5–7-1 Fujishirodai, Suita, Osaka 565, Japan. E-mail: minamino{at}ri.ncvc.go.jp
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, interleukin-1ß, and dexamethasone elevated AM secretion,
whereas transforming growth factor-ß1 and interferon-
suppressed
it in these three fibroblasts. Swiss 3T3 cells were shown to express
receptors specific for AM by both cAMP production and receptor binding
assay, and AM was found to stimulate DNA synthesis of quiescent cells
through the cAMP-mediated pathway. AM secreted from Swiss 3T3 cells was
also confirmed to augment cAMP production and DNA synthesis in the
cells themselves. These effects were inhibited by a neutralizing
monoclonal antibody against AM. These findings raise the possibility
that AM functions as a growth regulator in the case of Swiss 3T3 cells.
As AM receptors are widely distributed, AM secreted from fibroblast may
play a role as a local regulator in mesenchymal cells of inflammatory
or wounded regions. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our systematic survey has demonstrated that VSMC and EC actively produce and secrete AM and that their gene transcription levels are much higher than that of adrenal gland (10, 11). Among the substances examined, tumor necrosis factor (TNF), interleukin-1 (IL-1), and lipopolysaccharide (LPS) potently stimulated AM production in VSMC and EC (12, 13), suggesting that AM may participate in inflammation as well as in induction of hypotension in septic shock. In fact, the plasma AM concentration was markedly elevated in LPS-injected rats and in patients with septic shock (14, 15, 16). In LPS-injected rats, AM messenger RNA was detected and elevated in almost all tissues (14). These data indicate that vascular cells are major sites of synthesis and secretion of AM in septic shock model rats. However, nonvascular cells of some other tissues, such as lung, were deduced to be able to synthesize AM.
Fibroblasts of mesenchymal tissue are one of the important targets of inflammatory cytokines and LPS (17, 18). Recent observations have clarified that fibroblasts can secrete a variety of immunoregulatory cytokines and chemical mediators when they are stimulated with inflammatory cytokines or LPS (18). In heart tissue, for instance, fibroblasts are reported to support the development of cardiac hypertrophy and myocardial fibrosis when stimulated with angiotensin II (19). Factors secreted from human fibroblasts are shown to down-regulate the production of plasminogen activator inhibitor type 1 in cultured human EC (20). These data suggest that fibroblasts have potential functions not only in maintaining tissue integrity, but also in physiological regulation of parenchymal cells. In the present paper, we report active production of AM in human and mouse fibroblasts as well as regulation of AM production in these cells. Furthermore, we raise the possibility that AM functions as a growth regulator of Swiss 3T3 cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and bovine basic fibroblast growth factor
(FGF) were products of Boehringer Mannheim (Mannheim, Germany). Human
recombinant epidermal growth factor (EGF) was obtained from Austral
Biologicals (San Ramon, CA), and rat interferon- (IFN) and
genistein were obtained from Calbiochem (San Diego, CA). Human TNF
and IL-1ß were obtained from R&D Systems (Minneapolis, MN), and human
IFN was purchased from Pepro Tech (Rocky Hill, NJ). Forskolin was
obtained from Wako Pure Chemicals (Osaka, Japan), and NOC-18
[2,2'-(hydroxynitrosohydrazino)bis-ethanamine] was
purchased from Dojindo (Kumamoto, Japan). Escherichia coli
LPS (serotype 026:B6) was purchased from Paesel+Lorei (Frankfurt,
Germany), and NG-nitro-L-arginine
methyl ester was a product of Biomol Research Laboratory (Plymouth
Meeting, PA). Triton X-100 and Triton X-305 were obtained from Nacalai
Tesque (Kyoto, Japan). H-89
(N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide)
and H-7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine] were products
of Seikagaku Corp. (Tokyo, Japan). Human (h) AM-(40–52), its
N-Tyr derivative, and hAM-(46–52) were synthesized by a
peptide synthesizer (Applied Biosystems 431A, Foster City, CA) and
purified by reverse phase HPLC. Mouse (m) AM was purchased from Phoenix
Pharmaceutical (Mountain View, CA). Other peptides used in this study
were of human or rat (r) origin and were obtained from Peptide
Institute (Osaka, Japan). Dexamethasone was first dissolved in ethanol,
and then diluted with an incubation medium (DMEM containing 1% FCS).
T3 was first dissolved in 0.1 M NaOH, then
diluted with the incubation medium. Other substances were dissolved
according to the producer’s manuals and diluted with the incubation
medium.
Cell culture
Swiss 3T3 cells (mouse embryonic fibroblast) and Hs68 cells
(human newborn foreskin fibroblast) were maintained in DMEM containing
10% FCS (Filtron, Victoria, Australia) at 37 C in a humidified
atmosphere containing 5% CO2. NHLF cells (normal human
adult lung fibroblast), obtained from Clonetics Corp. (San Diego, CA),
were maintained in modified MCDB202 containing 2% FCS, 1 ng/ml basic
FGF, and 5 µg/ml insulin at 37 C in a humidified atmosphere
containing 5% CO2.
Peptide iodination
The N-Tyr derivative of hAM-(40–52), rAM, and a
methionine sulfoxide form of hAM were radioiodinated by the
lactoperoxidase method (21). N-Tyr-monoiodinated
hAM-(40–52) and a monoiodinated form of rAM with biological activity
were isolated by reverse phase HPLC using a linear gradient elution of
CH3CN from 10–60% in 0.1% trifluoroacetic acid (TFA) and
used as tracers for RIA and receptor binding assay, respectively. A
monoiodinated methionine sulfoxide form of hAM was also isolated by the
same procedures.
Measurement of immunoreactive (IR-) AM secreted from fibroblasts
and cellular IR-AM
Fibroblasts, grown to confluence in a six-well plate, were
incubated with an incubation medium (DMEM-1% FCS) containing
stimulants at 37 C for 14 h in a humidified atmosphere containing
5% CO2. The viability of fibroblasts after 12-h incubation
was estimated by trypan blue exclusion assay, and more than 95% of the
cells were viable under the present conditions. Culture media were
acidified with acetic acid (final concentration, 0.25 M),
and Triton X-100 was added (final concentration, 0.002%). The
resulting solution was heated at 100 C for 15 min and lyophilized. The
lyophilizates were dissolved in a RIA standard buffer and submitted to
RIA for AM. Details for the RIA system using antiserum 172-CI-7 against
hAM-(40–52) have been reported by Sakata et al. (21), and
the detection limit of this RIA system was 1 fmol/tube. An antiserum
172-CI-7, donated by Dr. Kitamura of Miyazaki Medical College, strictly
recognizes the C-terminal amide structure common to hAM, rAM, and pig
AM. Synthetic mAM, prepared based on the amino acid sequence recently
reported by Okazaki et al. (22), showed an affinity for the
antiserum equivalent to that of hAM. For measurements of cellular
IR-AM, fibroblasts were washed twice with PBS, scraped in 1
M acetic acid, and then collected. After heating at 100 C
for 10 min, the cell lysates were sonicated and centrifuged, and the
resulting supernatants were condensed with Sep-Pak C18
cartridges (Millipore Corp., Waters Division, Milford, MA), as reported
previously (14). The eluted materials were lyophilized and subjected to
RIA for AM.
Characterization of IR-AM in culture medium of fibroblasts
After 14-h incubation, conditioned culture media of the three
fibroblasts, Swiss 3T3, Hs68, and NHLF cells, without stimulation and
that of NHLF cells with stimulation of 10-7 M
dexamethasone were collected separately and acidified with acetic acid
(final concentration, 0.1 M). The peptide fraction of the
culture medium was desalted and condensed with a Sep-Pak
C18 ENV cartridge, as reported previously (14). After
lyophilization, the condensate was subjected to gel filtration on
Sephadex G-50 column (fine, 1 x 100 cm; Pharmacia, Uppsala,
Sweden). The peak fraction of IR-AM on the gel filtration was separated
by reverse phase HPLC on a Chemcosorb 5ODS-H (300 Å; 4.6 x 250
mm; Chemco, Osaka, Japan) using a linear gradient elution of
CH3CN from 10–60% in 0.1% TFA over 60 min at a flow rate
of 1 ml/min. An aliquot of each fraction was submitted to RIA for
AM.
cAMP production assay
Fibroblasts, grown to confluence in a 24-well plate, were washed
once with 0.5 ml DMEM, then preincubated in 25 mM
HEPES-buffered DMEM (pH 7.4) containing 0.01% BSA and 0.5
mM isobutylmethylxanthine (Nacalai Tesque) for 1 h.
The media were then replaced with the same buffer containing various
concentrations of rAM, hAM, hCGRP, their antagonists, other related
substances, and insulin, and the cells were further incubated at 37 C
for another 1–6 h. Aliquots of culture media were collected and
succinylated with succinic anhydride, as previously reported (23). The
resulting solution was lyophilized, dissolved in the RIA standard
buffer for the cAMP assay, and submitted to RIA of cAMP. The RIA was
performed as previously reported (23). The antiserum against cAMP was
donated by Dr. Kitamura.
Receptor binding assay
Fibroblasts, grown to confluence in a six-well plate, were
washed twice with 1 ml 20 mM HEPES-buffered DMEM (pH 7.4),
and incubated with various concentrations of 125I-labeled
rAM ([125I]rAM) in 1 ml DMEM containing 0.05% BSA and
0.01% Triton X-305 at room temperature for 1 h. Nonspecific
binding was determined in the presence of 2 µM unlabeled
rAM. In the competition studies, peptides [rAM, hAM, hAM-(22–52),
hCGRP, hCGRP-(8–37), and rat amylin (rAMY)] ranging from
10-11–10-6 M were added to the
incubation medium with [125I]rAM. After incubation, the
cells were washed once with ice-cold PBS and solubilized with 0.5
M NaOH. Radioactivity was measured with a -counter
(ARC-1000, Aloka, Tokyo, Japan). In the equilibrium-saturation
experiments, 1.85 x 10-11 M
[125I]rAM was added as a tracer, and the concentration of
unlabeled peptide was varied from 6.5 x 10-12 to
1.6 x 10-8 M. Binding data were analyzed
by nonlinear regression using a one- or two-site binding program
(GraphPad Prism, GraphPad Software, San Diego, CA) to calculate the
dissociation constant (Kd) and the maximum number of
binding sites (Bmax).
DNA synthesis assay
DNA synthesis assay was performed mainly according to the method
of Withers et al. (24). Swiss 3T3 cells were plated out in
six-well plates at 105 cells/well and incubated under
normal culture conditions. The cells were used for the assay after 7
days when they were confluent and quiescent. The cells were washed
twice with DMEM and incubated with AM and other substances in
DMEM-Waymouth’s medium (1:1, vol/vol) containing 0.05 mM
isobutylmethylxanthine, 0.01% BSA, and
5-[125I]iodo-2'-deoxyuridine ([125I]DU; 0.2
µCi/ml; Amersham, Aylesbury, UK). After 40-h incubation, the
fibroblasts were washed twice with ice-cold PBS and incubated in 5%
trichloroacetic acid for 30 min at 4 C. Trichloroacetic acid was then
removed, and the cells were washed twice with ethanol.
[125I]DU incorporated into nuclei was extracted with 1 ml
0.1 M NaOH containing 2% Na2CO3
and 1% SDS. Radioactivity recovered from cell nuclei was measured with
a -counter.
Neutralizing monoclonal antibody against AM
Monoclonal antibody against human AM-(46–52) (mAb-C1) was
prepared as follows. Synthetic human AM-(46–52), a carboxyl-terminal
peptide common to hAM and rAM, was conjugated to bovine thyroglobulin
(Sigma) by the carbodiimide method. The conjugate was emulsified with
Freund’s complete adjuvant, and the emulsion containing 4.7 µgEq of
the peptide was sc immunized to seven female BALB/c mice (5 weeks old)
eight times at 3-week intervals. Three days before cell fusion, the
emulsion was ip injected into the mouse with the highest titer. Fusion
of spleen cells from the immunized mouse with mouse myeloma cells,
X63-Ag8.653, was performed in a ratio of 5:1 using polyethylene glycol
4000 (Merck, Darmstadt, Germany) and a method previously reported (25).
Hybridomas were screened first for their ability to produce antibody
and second by the displacement test. The hybridomas selected by these
tests were cloned by limited dilution and ip injected to BALB/c mice.
Isotyping of mAb-C1 was carried out by the Ouchterlony technique (mouse
monoclonal antibody isotyping kit, Amersham). Binding affinity was
determined by a Scatchard plot analysis using a monoiodinated
methionine sulfoxide form of hAM. The IgG fraction of mAb-C1 (mAb-IgG)
was purified from mouse ascites using the Affi-Gel Protein A MAPS II
Kit (Bio-Rad, Hercules, CA), and the IgG fraction of mouse nonimmune
-globulin (NI-IgG) was purified from mouse -globulin (Organon
Teknika, Durham, NC) using the same method. For the neutralizing
experiments with AM, 10-8 M hAM or partially
purified IR-AM was incubated at 4 C for 24 h in the absence or
presence of mAb-IgG or NI-IgG before the cAMP production assay or the
DNA synthesis assay.
Partial purification of AM from culture medium of Swiss 3T3
cells
Conditioned media (250 ml) of Swiss 3T3 cells with or without
stimulation with TNF (50 ng/ml) were collected from 50 10-cm dishes
after 24-h incubation under normal culture conditions. Conditioned
media as well as plain incubation medium (250 ml) were acidified with
acetic acid (final concentration, 0.1 M). Peptide fractions
of the media were condensed with Sep-Pak C18 ENV cartridges
and then separated by reverse phase liquid chromatography on an LC-SORB
SPW-C-ODS (Chemco) using a gradient elution of CH3CN from
10–60% in 0.1% TFA at a flow rate of 10 ml/min for 50 min to
separate AM from TNF. IR-AM fractions corresponding to authentic rAM
were collected and lyophilized. The lyophilizates were dissolved in 1
ml DNA synthesis assay medium and submitted to cAMP production assay as
well as DNA synthesis assay.
Statistical analysis
Values were expressed as the mean ± SEM.
Statistical analysis of the results was performed with a one-way ANOVA,
followed by a multiple comparison test (Dunnett’s test), and
P < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The carboxyl-terminal
seven-amino acid amide structure is completely conserved in these five
AMs, and the antibody 172-CI-7 used in this study strictly recognizes
the carboxyl-terminal sequence of hAM. In practice, synthetic mAM and
rAM elicited the same affinity to the antibody as that of hAM,
indicating that the RIA for hAM used in this study can be used for
measurements of mAM, rAM, and other mammalian AMs.
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)
(10).
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). More than 80% of the total IR-AM
secreted from Swiss 3T3 and Hs68 cells was eluted at a mol wt of 6K
corresponding to hAM (Fig. 2, A and B). In the case of NHLF cells, only
41% of total IR-AM secreted from NHLF cells was eluted at a mol wt of
6K, and 59% of IR-AM emerged at a mol wt of 2K (Fig. 2C). Fractions
37–39 (Swiss 3T3 cells and Hs68 cells) and 36–39 (NHLF cells) were
each analyzed by reverse phase HPLC (Fig. 2, D–F). IR-AM of mol wt 6K
obtained from these cell lines emerged at the elution times of rAM and
hAM, respectively. Recently, the amino acid sequence of mAM has been
deduced form its precursor complementary DNA (22). mAM is a 50-amino
acid peptide like rAM and has three homologous replacements compared
with rAM, as shown in Fig. 1
. IR-AM eluted at the retention time of rAM
in Fig. 2D was thought to be composed of mAM. Thus, 6K IR-AM secreted
from Swiss 3T3, Hs68, and NHLF cells was concluded to be derived from
mAM of 50 residues or hAM of 52 residues. On the other hand, about 60%
of IR-AM secreted from NHLF cells that eluted at the lower mol wt
region emerged as a single peak on reverse phase HPLC (data not shown).
This peak of IR-AM was deduced to be a carboxyl-terminal fragment of
AM, as antiserum 172-CI-7 recognized the carboxyl-terminal
seven-residue structure common to mammalian AMs determined to date, and
an AM fragment with comparable molecular properties was observed in the
extracts of porcine duodenum (26). Based on these data, IR-AM measured
in the present study was deduced to be exclusively derived from hAM or
mAM and not from the cross-reactivity of unrelated substances. The
ratio of higher and lower mol wt IR-AM in the culture medium of NHLF
cells was not significantly altered even after stimulation with
10-7 M dexamethasone for 14 h (data not
shown).
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Based on the previous data of AM synthesis and secretion from cultured
rVSMC (12, 27, 28), we administered 11 substances to fibroblasts and
examined their effects on AM secretion (Table 1). In the case of Swiss
3T3 cells, TNF most potently stimulated AM secretion and increased
IR-AM content in the culture medium to 291% of the control value (Fig. 3A). The effect of TNF was dose
dependent, and its ED50 was estimated to be 3–5 ng/ml.
IL-1ß and LPS weakly enhanced AM secretion, and basic FGF, the
effective growth factor for fibroblasts, increased IR-AM content in the
medium by 48%. Dexamethasone strongly and dose dependently stimulated
AM secretion from Swiss 3T3 cells to 263% of the control value (Fig. 3B). Thyroid hormone, T3, and thrombin elevated IR-AM
content in the medium of Swiss 3T3 cells by 17% and 38%,
respectively. TGFß1 was the most potent suppressor of AM secretion,
dose dependently decreasing IR-AM content in the medium to 33% of the
control value (Fig. 4A). IFN and
forskolin strongly and dose dependently reduced the IR-AM content in
the medium to 44% and 39% of the control value (Fig. 4, B and C).
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increased the IR-AM content
in the culture medium to 206% and 131% of the control value,
respectively. IL-1ß was the most potent stimulator of AM secretion
from both Hs68 and NHLF cells, elevating IR-AM content in the culture
medium to 215% and 150% of the control value. LPS augmented AM
secretion from Hs68 cells, but did not induce any effect on NHLF cells.
EGF and basic FGF reduced AM content in the medium of NHLF cells to
74% and 84% of the control value, but was ineffective in the case of
Hs68 cells. TGFß1, IFN, and thrombin were suppressors of AM
secretion from both Hs68 and NHLF cells. Thrombin, in particular,
markedly decreased IR-AM content in the media of Hs68 and NHLF cells to
25% and 8% of the control value, respectively. TGFß1 induced a
strong effect, decreasing IR-AM levels to 31–47% of the control
value, whereas the effects of IFN were relatively weak, reducing the
secretion to about 80% in these two cell lines. Dexamethasone slightly
increased IR-AM content in the media of both Hs68 and NHLF cells, but
T3 and forskolin did not alter IR-AM secretion in either
cell line.
Effects of AM, CGRP, and their antagonists on cAMP production in
fibroblasts
To characterize AM receptors expressed on fibroblasts, we first
measured cAMP production in the three cell lines used in this study, as
AM was known to induce its effect mainly through elevating the
intracellular cAMP level. We measured the extracellular cAMP
concentration in this study to examine large numbers of samples after
confirmation that an extracellular cAMP level was correlated with an
intracellular increase in cAMP concentration. The cAMP concentration in
the medium was measured after incubating the cells with various
concentrations of AM and CGRP in the presence or absence of their
antagonists (Fig. 5). In the case of
Swiss 3T3 cells, rAM and hAM dose dependently increased the cAMP
concentration with ED50 values of 4 x
10-10 and 2.5 x 10-9 M,
respectively. hCGRP also elevated the cAMP concentration dose
dependently, and its ED50 value was estimated to be
1.3 x 10-7 M, which was 52 times greater
than that of hAM. The maximal cAMP concentration (
11
pmol/105 cells·h) achieved with the agonist stimulation
was more than 100 times higher than the basal cAMP concentration (0.1
pmol/105 cells·h). In the presence of hAM-(22–52), an AM
receptor antagonist (29), the dose-response curve of hAM was shifted in
parallel to the high concentration side, and its ED50
increased 8.8-fold. In the presence of hCGRP-(8–37), a CGRP receptor
antagonist (30), however, the dose-response curve of hAM also shifted,
and the ED50 of hAM increased 8.4-fold, which was
comparable to that of hAM-(22–52). rAMY, another member of the CGRP
superfamily, weakly increased the cAMP level to 3 pmol/105
cells·h at 10-6 M.
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In addition to AM, CGRP, and AMY, we administered seven peptides, which were generally known to increase the intracellular cAMP concentration (31, 32, 33, 34, 35, 36, 37), to Swiss 3T3 cells at a concentration of 10-6 M. Among them, vasoactive intestinal peptide and adenylate cyclase-activating polypeptide have been reported to stimulate cAMP production in Swiss 3T3 cells (31, 32). Pituitary adenylate cyclase-activating polypeptide, vasoactive intestinal peptide, peptide histidine-isoleucine amide, and GH-releasing factor stimulated cAMP production in Swiss 3T3 cells and increased the cAMP concentration in the medium to 280%, 360%, 310%, and 370% of the control value, respectively, whereas angiotensin II, ACTH, and endothelin-1 did not alter it. However, the increase in cAMP concentrations that these peptides induced was less than 3% of that caused by hAM or rAM.
Receptor binding assay
We characterized AM receptors expressed on fibroblasts by the
receptor binding assay. Each of the three fibroblast cell lines was
incubated with [125I]rAM in the presence of increasing
concentrations of unlabeled rAM, hAM, hAM-(22–52), hCGRP-(8–37), and
rAMY. All unlabeled peptides competed with [125I]rAM for
binding to Swiss 3T3 cells in a dose-dependent manner (Fig. 6A). On the other hand, Hs68 and NHLF
cells did not have any specific [125I]rAM-binding sites,
and hAM did not alter [125I]rAM binding to the cells
(data not shown). In the case of Swiss 3T3 cells, rAM most potently
inhibited [125I]rAM binding, with a median inhibitory
concentration (IC50) of 6.0 x 10-10
M. Equilibrium-saturation analysis of binding of
[125I]rAM was performed to demonstrate saturation of the
binding sites and to determine their number and affinity (Fig. 6B).
Binding data were analyzed by nonlinear regression using a one- or
two-site binding program to calculate Kd and
Bmax under the assumption that radiolabeled ligand had
affinity to the receptor equivalent to that of the unlabeled peptide. A
two-site binding program showed a better regression curve for the
equilibrium-saturation curve. The Bmax values of the two
binding sites were 0.02 and 2.28 pmol/106 cells, and the
Kd values were 0.29 x 10-9 and 36.3
x 10-9 M with 12,000 and 1,370,000
sites/cell, respectively. hAM and hCGRP inhibited
[125I]rAM binding with IC50 values of
4.1 x 10-9 and 5.1 x 10-7
M, respectively, whereas hAM-(22–52) and hCGRP-(8–37)
inhibited its binding with IC50 values of 9.0 x
10-7 and 6.0 x 10-8 M,
respectively.
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). The maximal effect of hAM was
observed at 10-6 M, which corresponded to
24.7% of that induced with 10% FCS. When hAM was administered in the
presence of insulin, [125I]DU incorporation was further
enhanced to 233% of the insulin control value, and the maximal effect
of hAM, corresponding to 71.5% of that induced with 10% FCS, was also
observed at 10-6 M AM. The dose-response
curves of [125I]DU incorporation to Swiss 3T3 cells in
the absence and presence of insulin was parallel to that of cAMP
production stimulated with hAM (Fig. 5). Although hCGRP also stimulated
[125I]DU incorporation into Swiss 3T3 cells in the
presence and absence of insulin, the effect of hCGRP was weak and
corresponded to 33% of that induced with hAM at 10-7
M (data not shown). AM and the CGRP antagonists,
hAM-(22–52) and hCGRP-(8–37), dose dependently inhibited DNA
synthesis stimulated by hAM in the presence of insulin (Fig. 8), similar to that observed in the cAMP
production assay. H-89, an inhibitor specific for cAMP-dependent
protein kinase (PKA), reduced hAM-induced [125I]DU
incorporation to about 40% in the presence of insulin, whereas basal
[125I]DU incorporation was not altered with H-89 at the
same dose (Fig. 9). In addition,
genistein (tyrosine kinase inhibitor, 5 x 10-6
M), H-7 (protein kinase C inhibitor, 5 x
10-6 M),
NG-nitro-L-arginine methyl ester
(nitric oxide synthase inhibitor, 10-5 M), and
NOC-18 (nitric oxide generator, 10-5 M) were
added to the culture medium of Swiss 3T3 cells, but these reagents did
not alter [125I]DU incorporation induced by
10-8 M hAM and 1 µg/ml insulin. These
results indicate that DNA synthesis stimulated with AM is mediated
through the AM receptor and the cAMP-PKA pathway.
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subclass. This antibody equally recognized rAM and hAM and showed no
cross-reactivity (<0.01%) with hCGRP and hAMY. As the
carboxyl-terminal region of AM is known to be essential for exerting
biological activity, we checked whether mAb-C1 could neutralize the
activity of AM. mAb-C1 and mouse -globulin were purified by protein
A affinity chromatography, as crude ascites disturbed the DNA synthesis
assay of Swiss 3T3 cells. mAb-IgG dose dependently inhibited cAMP
production in Swiss 3T3 cells stimulated with 10-8
M hAM in a range of 1–10 µg/ml, whereas NI-IgG was
ineffective at the same concentrations (Fig. 10A). Moreover, DNA synthesis
stimulated with 10-8 M hAM in the presence of
insulin was completely inhibited by 10 µg/ml mAb-IgG, whereas it was
not inhibited by NI-IgG (Fig. 10B). Thus, mAb-IgG (10 µg/ml) was
verified to have sufficient ability to neutralize the action of
10-8 M hAM.
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as well as a plain incubation medium,
IR-AM fractions corresponding to rAM were partially purified and
separated from TNF by reverse phase liquid chromatography. DNA
synthesis assay was performed in the presence of insulin. The IR-AM
fraction obtained from the conditioned medium without TNF
stimulation increased [125I]DU incorporation to 156% of
that in the plain incubation medium control, and the fraction from the
medium with TNF stimulation elevated it to 215% (Fig. 11B). The IR-AM concentrations
administered were estimated by RIA to be about 1.0 x
10-9 and 1.4 x 10-10 M in
the cases of TNF-stimulated and nonstimulated conditioned media,
respectively. When the same preparations were submitted to cAMP
production assay, the IR-AM fraction from TNF-stimulated and
nonstimulated conditioned medium significantly increased cAMP
production of Swiss 3T3 cells to 6.70 and 1.57 pmol/105
cells·h, respectively (Fig. 11A). Furthermore, [125I]DU
incorporation enhanced by the IR-AM fraction from TNF-stimulated
conditioned medium was inhibited about 60% with 10 µg/ml mAb-IgG,
but not with NI-IgG, whereas basal [125I]DU incorporation
induced by the IR-AM fraction of the plain incubation medium was not
altered by the addition of 10 µg/ml of either mAb-IgG or NI-IgG (Fig. 11B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TNF, IL-1ß, and LPS, which are major factors in the induction of
septic shock and inflammation (39, 40), augmented AM secretion from
Swiss 3T3, Hs68, and NHLF cells, except for LPS in NHLF cells.
Stimulation of these substances is comparable to or less than that
observed in the case of cultured rVSMCs (12). These results indicate
that factors that induce septic shock and inflammation generally
enhance AM synthesis and secretion from fibroblasts as well as VSMC and
EC (12, 13). IFN suppressed AM secretion from all of the cell lines
in a manner similar to that of EC and VSMC (13, 28), and TGFß1 also
reduced it, as in the case of EC (13). Dexamethasone was one of the
strongest stimulators of AM secretion in the three fibroblasts as well
as in VSMC and EC (13, 27). These data indicate that AM synthesis in
fibroblasts is principally regulated by a mechanism similar to that of
VSMC and EC, especially in the case of inflammatory cytokines, IFN
and glucocorticoid. In the other substances examined, several
discrepancies were observed between fibroblast cell lines and between
fibroblasts and VSMC. Basic FGF and T3 stimulated AM
synthesis in Swiss 3T3 cells, but basic FGF suppressed it in NHLF
cells, and T3 did not alter it in either Hs68 or NHLF
cells. EGF reduced AM synthesis in NHLF cells and was ineffective in
Swiss 3T3 and Hs68 cells. The largest differences were observed in the
cases of thrombin and forskolin. Thrombin stimulated AM synthesis in
Swiss 3T3 cells, but potently suppressed it in both Hs68 and NHLF
cells. Although we have to date failed to characterize the mechanism of
thrombin action not only in fibroblasts but also in EC and VSMC (13, 28), the different responses of each cell line might be elucidated in
the future through identification of the intracellular signal
transduction systems. Forskolin decreased AM synthesis in Swiss 3T3
cells, but did not alter it in Hs68 and NHLF cells. The different
actions of forskolin between cell lines may depend on receptors
expressed on the cells. The different effects observed in the three
fibroblast cell lines might be derived from the species or origin of
the cells.
This study also demonstrated that AM has specific receptors on fibroblasts and stimulates cAMP production. Especially, Swiss 3T3 cells of mouse embryo origin are found to express high affinity AM receptors based on the results of the cAMP production assay and the receptor binding assay. rAM showed a dissociation constant of 0.6 x 10-9 M in the binding assay when [125I]rAM was used as a tracer and an ED50 of 0.4 x 10-9 M in the cAMP production assay. These values are comparable to each other and are relatively low for biologically active peptides, indicating that binding and intracellular events are induced by the same specific mechanism. Swiss 3T3 cells have been reported to express AM receptor, but not CGRP receptor (24), and to date, one AM receptor has been cloned and sequenced (41). In these reports, AM receptor was shown not to respond to CGRP or AMY even at a concentration of 10-6 M. The present study showed that CGRP stimulated cAMP production and inhibited [125I]rAM binding to Swiss 3T3 cells, and that its effect was weaker than that of AM. AMY elicited faint, but significant, effects in these two assays. Thus, AM is concluded to elicit its effect through receptors specific for AM, although we need to characterize the low affinity binding site for [125I]rAM.
For antagonists, [125I]rAM binding to Swiss 3T3 cells was more potently inhibited with hCGRP-(8–37) than with hAM-(22–52), but hCGRP showed less than 1% of the activity of hAM in the [125I]rAM binding assay. In the case of rat astrocytes, Zimmermann et al. reported data comparable to those of this study, i.e. hCGRP-(8–37) had higher affinity than hAM-(22–52) in the AM binding assay, but [125I]rAM binding was more potently inhibited with hAM than with hCGRP (42). In the cAMP production assay, hCGRP-(8–37) and hAM-(22–52) shifted a dose-response curve of hAM to a comparable extent, but the potency of hCGRP corresponded to a few percentages of that of hAM. On the other hand, Champion et al. reported that AM-(22–52) antagonized vasodilatory responses to CGRP, but not to AM, in the cat (9). These data indicate that hAM-(22–52) can be used as an antagonist for AM receptors, but is not a specific and appropriate one.
Among the seven examined peptides that were known to elevate the intracellular cAMP concentration, GH-releasing factor most potently increased cAMP production to 0.37 fmol/105 cells·h, 370% of the control value, but this level was only 3% of the cAMP concentration stimulated with hAM or rAM. This result indicates that AM is the most potent peptide in the cAMP production of Swiss 3T3 cells, suggesting that AM may have an important effect on Swiss 3T3 cells as an autocrine factor.
Hs68 and NHLF cells are found to express CGRP receptors rather than AM receptors, as the ED50 values of hAM for the cAMP production assay of Hs68 and NHLF cells were about 100 and 50 times larger than that of hCGRP. In the receptor binding assay, [125I]rAM did not show any specific binding to these two human fibroblast cell lines. Furthermore, maximal cAMP concentrations in the culture media of Hs68 and NHLF cells induced with hAM were 5–10% that in Swiss 3T3 cells. Although Hs68 and NHLF cells secrete AM at a rate 30 times higher than that of Swiss 3T3 cells, these data suggest that AM secreted from Hs68 and NHLF cells mainly functions not as an autocrine factor but as a paracrine factor, acting on other surrounding cells expressing AM receptors. On the other hand, it is another possibility that continual exposure to a high concentration of AM may down-regulate expression of the AM-specific receptor on Hs68 and NHLF cells to reduce the effect of AM. The lack of response of Hs68 and NHLF cells to forskolin in the AM production assay might be related to major expression of CGRP receptors and low expression of AM receptors, both of which are coupled with the adenylate cyclase system. As the vasodilatory effects of AM were shown by Nossaman et al. (8) to be quite different in each species, species differences should be taken into account to elucidate the different responses observed above.
We initiated a survey of biological functions of AM secreted from Swiss 3T3 cells, after we confirmed the synthesis and secretion of AM and the expression of AM-specific receptors on this cell line. Withers et al. reported that forskolin, 8-bromo-cAMP, as well as AM stimulated DNA synthesis and proliferation of quiescent Swiss 3T3 cells in the presence and absence of insulin (24, 38). In this study, we also confirmed that AM dose dependently stimulated DNA synthesis in quiescent Swiss 3T3 cells in the presence and absence of insulin, and that a maximal stimulation level of DNA synthesis in the presence of insulin corresponded to 71.5% of that achieved with 10% FCS, which was comparable to that reported for bombesin and platelet-derived growth factor (38). Because insulin does not increase cAMP production in Swiss 3T3 cells, the enhancement of DNA synthesis induced by insulin is not thought to be mediated via a cAMP-mediated pathway. The stimulatory effect of hAM on DNA synthesis was inhibited with hAM-(22–52) and hCGRP-(8–37) in a manner similar to that observed in the cAMP production assay. Furthermore, AM was shown to be the strongest stimulant of cAMP production in Swiss 3T3 cells among the biologically active peptides examined, and DNA synthesis augmented with AM was inhibited with H-89, a specific inhibitor of PKA. On the other hand, neither tyrosine kinase inhibitor nor protein kinase C inhibitor suppressed AM-induced DNA synthesis. Nitric oxide is reported to significantly contribute to the vasodilatory effects of AM, but the inhibitor of nitric oxide synthase and nitric oxide generator did not affect the DNA synthesis stimulated by AM. These results indicate that AM functions as a proliferative factor in the growth regulation of Swiss 3T3 cells, and that this effect is induced through the AM-specific receptor and the adenylate cyclase-PKA system.
The proliferation of VSMCs and mesangial cells is reported to be inhibited by AM through the cAMP-mediated pathway (43, 44). This is a sharp contrast to the growth stimulatory effect of AM on Swiss 3T3 cells. Miller et al. reported that AM also stimulated growth of the breast and lung cancer cell lines via the cAMP-PKA system (45). Among the types of PKA identified to date, PKA-RI is reported to inhibit and PKA-RII to stimulate the cell growth (46). Based on these data, it is deduced that differences in the types of PKA used in the intracellular signal transduction system result in opposite effects of AM on the cell growth.
To examine the possibility of whether AM secreted from Swiss 3T3 cells acts on them as a growth regulator, a neutralizing monoclonal antibody was prepared and used for the experiment. mAb-IgG (10 µg/ml) was confirmed to have the ability to neutralize the effect of hAM up to 10-8 M. As direct administration of mAb-IgG to the culture medium did not significantly alter [125I]DU incorporation into the cells, IR-AM secreted from Swiss 3T3 cells was partially purified, incubated with mAb-IgG, and then applied to the culture medium of Swiss 3T3 cells. DNA synthesis of Swiss 3T3 cells induced with endogenous IR-AM was inhibited by mAb-IgG, but not by NI-IgG. These results suggest that endogenous AM could function as a growth factor in Swiss 3T3 cells if the AM concentration was more than 10-9 M. It is highly feasible that secreted AM is accumulated, and its concentration in the intercellular space is increased to more than 10-9 M based on the secretion rate data. Moreover, mAM is expected to have affinity comparable to or higher than that of rAM for the AM receptors on Swiss 3T3 cells, as deduced from the amino acid sequences of mAM. Based on these data, it is possible that AM functions as a growth regulator in the case of Swiss 3T3 cells.
Owji et al. reported that the abundant and specific binding of [125I]rAM was observed in all of the rat tissues examined, including heart, lung, spleen, spinal cord, and skeletal muscle (47). Although it is necessary to identify these receptors, the wide distribution of AM receptors as well as AM synthesis in fibroblasts shown in this study indicate that AM may be involved in many types of cell-cell communications. Miller et al. reported that AM and its receptor are expressed in human tumor cell lines at a high ratio, and that neutralizing monoclonal antibody against AM inhibits tumor cell growth (45). They implicated the presence of an autocrine growth regulatory mechanism through AM and its receptor in neoplastic proliferation.
Montuenga et al. reported that AM and AM receptor are expressed throughout the organogenic stage of mouse and rat embryos, and that they are present not only in parenchymal cells of heart, placenta, and brain, but also in mesenchymal cells, including fibroblast of lung, skeletal, and integumentary tissues (48). We demonstrated that AM is actively secreted from fibroblasts and stimulates DNA synthesis in Swiss 3T3 cells of mouse embryo origin. These findings suggest that AM may act on embryogenesis as an autocrine or paracrine regulator. On the other hand, AM stimulated DNA synthesis in Swiss 3T3 cells, but not in Hs68 and NHLF cells, of newborn and adult human fibroblasts. The differences observed between these fibroblasts may come from the differences in the original tissues and in the stage of development, although the cells might have been transformed during culture.
Under normal conditions, tissues are maintained in a quiescent state
that allows only for the replacement of effete cells without
overgrowth. Many forms of injury can perturb the system and activate a
cascade of repair events leading to excessive connective tissue
formation (49). A repair sequence begins with rapid infiltration of
neutrophils and then macrophages, which secrete soluble mediators or
cytokines that trigger the proliferation of fibroblasts, ECs, and
VSMCs. TNF and IL-1ß secreted from leukocytes then stimulate AM
synthesis and secretion from fibroblasts in the wound region (50).
Studies of dermal wounds have shown that wound macrophages also produce
insulin-like growth factor I, which enhances cAMP-stimulated
mitogenesis in a manner similar to that of insulin (51). These data
suggest that AM secreted from fibroblasts may play an important role in
tissue repair with cytokines and insulin-like growth factor I.
In the patients with heart failure and renal failure, circulatory
levels of AM are reported to be higher than those in healthy volunteers
(52, 53). In these patients, plasma concentrations of inflammatory
cytokines, such as TNF and IL-1ß, are also known to be elevated
(54, 55). Although circulating AM is deduced to be mainly secreted from
VSMCs and ECs (12, 13), the data obtained in the present study indicate
that fibroblasts and mesenchymal tissues could be another major
candidate for secreting AM into the circulation in these patients.
In conclusion, we demonstrated that AM is synthesized and secreted from fibroblasts in a native and biologically active form. The synthesis and secretion of AM in fibroblast are regulated by inflammatory cytokines, such as TNF and IL-1, LPS, growth factors, and hormones in a manner similar to that of VSMC and EC. As Swiss 3T3 cells express receptors specific for AM, and AM stimulates DNA synthesis of quiescent fibroblasts, AM secreted from Swiss 3T3 cells is deduced to function as a growth regulator. These data suggest that AM may be a local regulator of cell growth and inflammation in the mesenchymal tissue in addition to its potent effect as a vasorelaxant in the vascular wall.
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Acknowledgments |
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Footnotes |
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Received October 14, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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vivo: analysis by mRNA phenotyping. Science 241:708–712 and their
inhibitors in undialyzed chronic renal failure, CAPD and hemodialysis
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) or presence of 1 µM hAM-(22–52)
(•) and 1 µM hCGRP-(8–37) (
), in addition to
increasing concentrations of rAM (
). B, Swiss 3T3 cells were
stimulated with increasing concentrations of hCGRP in the absence (
)
or presence of 1 µM of hAM-(22–52) (
) and 1
µM hCGRP-(8–37) (
). Each point
represents the mean ± SEM of four separate wells.
