© 1997 American Heart Association, Inc.
Articles |
Role of Activin-A and Follistatin in Foam Cell Formation of THP-1 Macrophages
From the Department of Geriatrics (K.K., M.A., M.E., M.Y., K.T., S.I., M.I., M.H., H.O., Y.O.) and the Third Department of Internal Medicine (N.Y.), Faculty of Medicine, and the Department of Molecular Biology and Medicine, Research Center for Advanced Science and Technology (T.K.), University of Tokyo, Tokyo, Japan.
Correspondence to Yasuyoshi Ouchi, MD, Department of Geriatrics, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan.
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Abstract |
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Abstract Macrophage (M
) foam cell formation is a
characteristic event that occurs in the early stage of
atherosclerosis. To examine the roles of activin-A, a
member of the transforming growth factor-ß superfamily, and
follistatin, the binding protein for activin-A, in M function, we
investigated their effects on foam cell formation of THP-1 Ms. When
THP-1 Ms were treated with activin-A (5 nmol/L), foam cell formation
and cellular cholesteryl ester accumulation were decreased. This
downregulation was paralleled by a reduction in cell association
and degradation of acetylated LDL. The inhibitory
effect of activin-A on cell association and degradation was dose
dependent, and the effect was blocked by concomitant addition of
follistatin. Activin-A (5 nmol/L) also decreased the Bmax for
acetylated LDL and scavenger receptor mRNA expression.
Follistatin showed an effect opposite to that of activin-A and promoted
M foam cell formation and cellular cholesteryl ester accumulation.
It increased binding, cell association, and degradation of
acetylated LDL and upregulated scavenger receptor mRNA
expression. Because follistatin is the binding protein for activin-A,
follistatin's effect is considered to be mediated by blocking the
inhibitory effect of intrinsic activin-A. These results
indicate that activin-A inhibits and follistatin promotes M foam
cell formation by regulating scavenger receptor mRNA expression. We
conclude that activin-A and follistatin play important roles in the
process of atherosclerosis by regulating M foam
cell formation.
Key Words: activin-A • follistatin • scavenger receptor • foam cell formation
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Foam cell formation of macrophages is observed in the early stage of atherosclerosis.1 M
s scavenge
lipoproteins that have undergone oxidative modification in the vascular
wall. Ms take up modified LDLs via the SR pathway. Unlike the LDL
receptor, the SR is not downregulated by cellular
cholesterol content; consequently, Ms become foam cells
when they take up excessive amounts of modified lipoproteins via the
SR.2 It has been shown that several cytokines and
factors regulate SR expression and/or activity. M-CSF is a positive
regulator that increases the activity of the SR.3 4 5
Lipopolysaccharide,6 tumor necrosis
factor-
,6 7 TGF-ß,8
interferon-
,9 10 and GM-CSF11 are negative
regulators.
Activin-A, a member of the TGF-ß superfamily,12 13 has a
variety of important biological functions: it promotes secretion of
follicle-stimulating hormone from anterior pituitary
cells,14 induces differentiation of erythroid
cells,12 promotes survival of neuronal
cells,15 and induces mesoderm in Xenopus during
development.16 Follistatin is a 35-kD
glycoprotein that binds to activin and neutralizes its
effect.17 18 19 Follistatin is widely expressed in the
body20 21 and is considered to be an important modulator
of activin. We previously demonstrated that activin-A is expressed in
arteriosclerotic lesions of the Watanabe heritable
hyperlipidemic rabbit.22 We also showed
that follistatin is expressed in the neointima and media of
the rat denuded carotid artery and is produced by vascular
SMCs.23 Although it has been reported that activin-A
inhibits vascular endothelial cell
growth24 and promotes SMC growth,25 nothing
has been reported about the effect of activin-A and follistatin on the
function of M, which are key regulators of atherogenesis. In the
present study, we examined the effects of activin-A and follistatin
on M function and found that the transformation of THP-1 M into
foam cells was inhibited by activin-A and promoted by follistatin. We
also revealed that this regulation may take place at the mRNA level of
the SR. Together with the existence of these factors in atherosclerotic
lesions, our results suggest that activin-A and follistatin may play
important roles in the development of
atherosclerosis.
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Methods |
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Materials
Recombinant human activin-A was provided by Dr Y. Eto of the Central Research Laboratory of Ajinomoto Co, Ltd. Activin-A was purified from the media of Chinese hamster ovary cells bearing an expression vector for the cDNA of the ßA subunit of activin.26 It was confirmed that purified activin-A was free of endotoxin. Recombinant human follistatin was also provided by Dr Y. Eto. Recombinant human M-CSF was obtained from Morinaga Milk Industry Co, Ltd. The following materials were purchased from various sources: human platelet-derived growth factor-BB from Genzyme; [
-32P]dCTP and [125I]NaI from NEN
Research Products; RPMI 1640 from GIBCO BRL; fetal bovine serum
from Bioserum; and TPA from Sigma Chemical Co. All other chemicals were
of analytical grade.
Cell Culture
THP-1 cells from the American Type Culture Collection
(Rockville, Md) were cultured in RPMI 1640 medium supplemented with 5%
fetal bovine serum, 1.5 g/L NaHCO3, 80 mg/mL
kanamycin monosulfate, and 15 mmol/L HEPES (pH 7.4). The
cells were maintained in a humidified 5% CO2 chamber at a
cell density of 2 to 10 x105/mL. THP-1 cells were
differentiated in RPMI 1640–5% fetal bovine serum medium containing
100 ng/mL TPA for 48 hours. After being washed three times with
PBS, differentiated THP-1 cells, termed THP-1 M, were cultured in
RPMI 1640–5% LPDS medium in the presence of activin-A or follistatin
for 24 to 48 hours.
Preparation of Ac-LDL, Lipoprotein-Deficient Serum, and
125I-Labeling of Ac-LDL
Plasma was collected from normolipidemic volunteers in a fasting
state. LDL was isolated from the plasma by sequential density
ultracentrifugation.27 The fraction of
d>1.21 g/mL was collected and dialyzed extensively
in sodium phosphate buffer (150 mmol/L NaCl and 2
mmol/L sodium phosphate, pH 7.4). It was sterilized by
filtration (0.45 µm) and used as LPDS. LDL was
acetylated by repetitive additions of acetic anhydride as
described previously.28 Ac-LDL was
radioiodinated with [125I]NaI by the iodine
monochloride method.29
Oil Red O Staining
After THP-1 Ms (1.6x106 cells per well in
six-well plates) were cultured with activin-A (5 nmol/L) or
follistatin (500 ng/mL) for 48 hours, cells were incubated in
RPMI 1640–5% LPDS medium containing 50 µg/mL Ac-LDL in the
presence of activin-A (5 nmol/L) or follistatin (500
ng/mL) at 37°C for 24 hours. After being washed three times
with PBS, cells were fixed with 6%
paraformaldehyde–0.1 mol/L sodium phosphate (pH
7.4) and stained with oil red O and hematoxylin. To evaluate the extent
of foam cell formation, the red-stained areas were quantified by using
NIH Image 1.56. In brief, the red-stained areas were "extracted"
from the original image (x400 magnification). Then their sizes were
calculated and divided by the size of the entire original area (cell
area was almost the same in all observed fields). This analysis
was performed in four random fields for control, activin-A–treated,
and follistatin-treated cells.
Measurement of CE
After THP-1 Ms (2.0x106 cells per well in
six-well plates) were cultured with activin-A (5 nmol/L),
follistatin (500 ng/mL), or fucoidan (100 µg/mL) for 48
hours, cells were incubated in RPMI 1640–5% LPDS medium containing 50
µg/mL Ac-LDL in the presence of activin-A (5 nmol/L),
follistatin (500 ng/mL), or fucoidan (100 µg/mL) at
37°C for 36 hours. After the cells were washed three times with TBS
(pH 7.4) containing 2 mg/mL bovine serum albumin and
another three times with TBS, cellular lipids were extracted in 2 mL
hexane/isopropanol (3:2, vol/vol). The extracts were evaporated
under a stream of N2 gas and resolved in 150 µL
isopropanol. Total and free cholesterol values were
quantified by a fluorimetric enzymatic method using Determiner TC 555
and Determiner FC 555 (Kyowa Medics). The difference between the
amounts of total and free cholesterol was determined as
cellular CE content. Each value was corrected with respect to the
amount of cell protein that was measured by the Bio-Rad protein assay
(Bio-Rad Labs).
Cell Association, Degradation, and Binding of
125I–Ac-LDL
THP-1 Ms (1x106 cells per well in 12-well
plates) were cultured with activin-A (5 nmol/L) or follistatin
(500 ng/mL) for 48 hours. After being washed three times with
PBS, cells were incubated in RPMI 1640–5% LPDS medium containing
125I–Ac-LDL at 37°C for 6 hours (to measure cell
association and degradation) or at 0°C for 2 hours (to measure
binding) as described previously.30 Each value was
corrected with respect to the amount of cell protein. Nonspecific cell
association, degradation, and binding were determined by adding a
40-fold excess of unlabeled Ac-LDL. Specific values were calculated by
subtracting the nonspecific value from the total value.
RNA Extraction and Northern Blot Analysis
Total RNA was extracted by the acid guanidinium
thiocyanate/phenol/chloroform method31 from THP-1
Ms that had been cultured with activin-A (5 nmol/L) or
follistatin (500 ng/mL) for 24 hours. Poly(A)+ RNA
was prepared by using Oligo-dT30 "Super" (Nippon Roche) according
to the manufacturer's protocol. Then, 4 µg poly(A)+ RNA
was subjected to electrophoresis in a 1% formaldehyde/agarose gel and
transferred to a nylon membrane (Gene-Screen Plus, NEN Research
Products). The membrane was hybridized with a randomly primed,
32P-labeled human M SR cDNA probe32 or with
a human ß-actin cDNA probe (Wako Pure Chemicals). The membrane was
washed in 15 mmol/L NaCl, 1.5 mmol/L sodium
citrate, and 0.1% SDS solution at 65°C for 20 minutes and then
exposed to x-ray film at -80°C for 2 days. The signal density of SR1
and SR2 was measured by densitometry, and values were corrected by
comparison with the value for the density of ß-actin.
Statistics
All values in the text and figures are expressed as mean±SEM.
The data were analyzed by one-factor ANOVA. If statistical
significance was found, the Newman-Keuls test was performed to isolate
the difference between groups. A value of P<.05 was
considered significant.
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Results |
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Foam Cell Formation of THP-1 M
sWhen THP-1 M
s were incubated with Ac-LDL (50
µg/mL) for 24 hours, lipid droplets accumulated within the
cytoplasm (Fig 1A
, red stained area). Compared with control THP-1
Ms, activin-A suppressed and follistatin enhanced transformation of
foam cells (Fig 1B).
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CE Content in THP-1 Ms
Because cellular CE accumulation is known to parallel M foam
cell formation,2 we examined the effects of activin-A and
follistatin on cellular CE content. CE mass after a 36-hour incubation
with 50 µg/mL Ac-LDL was 14.9±1.9 µg/mg cell protein
in control THP-1 Ms (Fig 2). Activin-A
decreased CE content to 11.0±0.7 µg/mg cell protein (74% of
control). Fucoidan, which is a competitive inhibitor of
Ac-LDL, also markedly decreased cellular CE content. On the other hand,
follistatin increased cellular CE content to 18.5±0.8 µg/mg
cell protein (124% of control). This effect was almost equivalent to
that of anti–activin-A antibody (data not shown).
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Cell Association and Degradation of 125I–Ac-LDL
To clarify the mechanisms of regulation of foam cell formation by
activin-A and follistatin, we examined their effects on the activity of
the SR. As shown in Fig 3 activin-A
decreased cell association and degradation of 125I–Ac-LDL
in a dose-dependent manner. Although the inhibitory effect
of activin-A was less potent than that of TGF-ß, activin-A (>5
nmol/L) significantly decreased SR activity. The
inhibitory effect of activin-A (5 nmol/L) was
abolished when follistatin (500 ng/mL) was added concomitantly.
Because follistatin is the binding protein for activin-A, this result
confirms that the inhibition of SR activity was indeed caused by
activin-A. Follistatin at concentrations >100 ng/mL
significantly increased cell association and degradation of
125I–Ac-LDL (Fig 4). The
effect of follistatin was almost as strong as that of M-CSF, which is
known to increase activity of the SR.
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Binding of 125I–Ac-LDL
Next we investigated the effects of activin-A and follistatin on
the binding of Ac-LDL. As shown in Fig 5, all binding curves showed saturation. Activin-A (5 nmol/L)
decreased and follistatin (500 ng/mL) increased the binding of
125I–Ac-LDL. Scatchard analysis revealed that Bmax
was decreased by activin-A and increased by follistatin (Bmax=60, 38,
and 79 ng/mg cell protein for control, activin-A–treated, and
follistatin-treated cells, respectively). On the other hand, the
binding affinity for Ac-LDL was not altered
(Kd=120 µg/mL).
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Northern Blot Analysis for SR mRNA Expression
To clarify the mechanisms of this downregulation/upregulation of
Ac-LDL binding, we examined expression of the SR at the mRNA level.
THP-1 Ms expressed two types of SR mRNA, 5.5-kb SR1 and 4.4-kb SR2.
Activin-A slightly decreased SR1 and clearly decreased SR2 mRNA
expression to 80% and 40% of control levels, respectively (Fig 6). In contrast, both SR1 and SR2 mRNA
expression was markedly increased by follistatin to 310% and 170% of
control levels, respectively.
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Discussion |
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Activin-A, first identified as an activating factor for the release of follicle-stimulating hormone from anterior pituitary cells, is now known to be expressed widely in the body and is recognized as a regulator of diverse important cellular functions.33 In vascular wall cells, activin-A has been shown to inhibit endothelial cell growth24 and promote SMC growth.25 In the present study, we have shown that activin-A also had an effect on M
s, which play a major role in the
atherosclerotic process.
Our results revealed that activin-A inhibited foam cell formation of
and CE accumulation in THP-1 Ms. This downregulation was
paralleled by a reduction in cell association and degradation of
Ac-LDL. Because it is known that CE in foamy Ms is derived from
cholesterol released from degraded lipoprotein
particles,2 our results suggest that inhibition of foam
cell formation and CE accumulation may be mediated by reductions in the
cell association and degradation of Ac-LDL. This reduction is possibly
caused by a decrease in the number of SRs, because the binding study
showed that activin-A decreased Bmax, whereas it had little influence
on affinity.
Which process yields the reduction in SR expression/activity by activin-A? The possibility that activin-A reduces Ac-LDL binding by directly interfering with ligand binding can be excluded, because in the cell association and degradation study, cells were treated with activin-A only before Ac-LDL was added. Furthermore, if activin-A were a competitive inhibitor, it could not suppress SR mRNA expression. Therefore, it is unlikely that activin-A acted as a competitive inhibitor.
Previous studies have established that lipopolysaccharide can
inhibit M scavenger function by blocking ligand binding and by
decreasing receptor gene expression. To rule out the possibility that
the purified activin-A that we used in the present study was
contaminated with lipopolysaccharide, we measured the level of
endotoxin in activin-A solution by a turbidimetric time
assay.34 As a result, endotoxin was undetectable (<10
pg/mL). Therefore, it is unlikely that the reduction in SR
expression/activity was caused by the concurrent presence of
lipopolysaccharide with activin-A.
With respect to the reduced binding capacity for Ac-LDL, we also found that activin-A downregulated the SR at the mRNA level. However, the precise mechanism of this downregulation (eg, instability, transcriptional downregulation, or the effect of another protein) is unknown. Taken together, our data suggest that the inhibition of foam cell formation by activin-A is regulated, at least in part, by downregulation of the SR at the mRNA level.
The inhibitory effect of activin-A was modest compared with
that of TGF-ß (Fig 3
). Previously, Bottalico et al38 had
shown that TGF-ß inhibited Ac-LDL degradation by 
70%. They also
showed that 24 pmol/L TGF-ß was sufficient to exert the
maximum effect for suppression of Ac-LDL degradation. Activin-A and
TGF-ß bind to heteromeric receptor complexes known as type I and type
II receptors. The type II receptor determines ligand-binding
specificity, and each receptor interacts with a distinct repertoire of
type I receptor.35 Therefore, even though the
inhibitory effect of activin-A is less potent than that of
TGF-ß, the functional roles of activin-A and TGF-ß are considered
to be different.
Follistatin is the binding protein of activin-A and is known to inhibit
its action.17 18 19 In the present study, we have shown
that follistatin itself promotes M foam cell formation and CE
accumulation. Follistatin also increased the activity and expression of
the SR. Because it is already shown that THP-1 Ms can produce
activin-A,12 36 these effects of follistatin may result
from blockade of the effect of intrinsic activin-A. Indirectly, the
evidence that enhancement of CE accumulation by follistatin was almost
equivalent to that by anti–activin-A antibody indicates that
follistatin acted as a blocker of intrinsic activin-A. When THP-1 cells
and human monocytes are induced to differentiate, both types of cell
come to express the SR37 38 39 40 and produce
activin-A.12 36 41 42 Because we showed that activin-A
decreased SR expression, it is suggested that basal expression of SR is
kept suppressed by simultaneously produced activin-A during
M differentiation.
Several lines of evidence indicate that activin-A and follistatin are
involved in atherogenesis. We found that activin-A is expressed in
arteriosclerotic lesions of Watanabe heritable
hyperlipidemic rabbits.22 Activin-A is
predominantly expressed in the neointima of the diseased
artery. In vitro experiments from other groups have shown that
activin-A is produced by human monocytes/Ms41 42 as
well as THP-1 Ms.12 36 On the other hand, follistatin
is expressed in the neointima and media of the rat denuded
carotid artery.23 Furthermore, follistatin expression is
increased in atherosclerotic lesions in comparison with normal vascular
tissue. In vitro experiments have suggested that SMCs can produce
follistatin.23 Although it is not known how activin-A and
follistatin are differently regulated in
arteriosclerotic lesions, the balance of these
factors may determine the level of SR expression on Ms.
In conclusion, we demonstrated that activin-A inhibited the expression
and activity of the SR in THP-1 M, resulting in suppression of
cellular CE accumulation and M foam cell formation. We also revealed
that follistatin, probably by blocking the inhibitory
effect of intrinsic activin-A, increased expression and activity of the
SR, thus promoting accumulation of cellular CE and the transformation
of Ms into foam cells. Activin-A and follistatin, which are
expressed in arteriosclerotic lesions, may regulate
the M foam cell formation that is a key step in the process of
atherosclerosis.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported in part by a grant-in-aid (No. 08670771) for Scientific Research from the Ministry of Education, Science and Culture of Japan. We would like to thank Dr Y. Eto of the Central Research Laboratory of Ajinomoto Co, Ltd, for providing us with activin-A and follistatin and giving us useful advice. We also thank Masae Watanabe and Hitomi Yamaguchi for excellent technical assistance.
Received November 8, 1996; accepted April 28, 1997.
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