Original Paper |
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Acta Biochim Biophys |
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doi:10.1111/j.1745-7270.2006.00222.x |
Effects of Raloxifene on Caveolin-1 mRNA and Protein Expressions in Vascular
Smooth Muscle Cells
Fa-Lin YANG1#*, Hong HE2#, Xian-Xi LIU1*, Bing TU1, Xian-Wei ZENG3, Ji-Xin SU1, Xin WANG2, and Qin HU2
1 Institute of Biochemistry and Molecular Biology,
Medical College, Shandong University, Jinan 250012, China;
2 Department of Cardiology, Qilu Hospital, Shandong
University, Jinan 250012, China;
3 Department of Clinical Medicine, Weifang Medical
College, Weifang 260041, China
Received: May 23,
2006
Accepted: August 9,
2006
This work was supported
by the grants from the Grant-in-Aid for Scientific Research of the Ministry of
Education, China (No. 2004.527), the Grant-in-Aid for China-Japan Sasagawa
Researchers of the Ministry of Health, China (No. 083), and the Natural Science
Foundation of Shandong Province, China (No.Y2005C50)
# These authors
contributed equally to this work
*Corresponding
authors:
Fa-Lin YANG: Tel,
86-531-82169436; Fax, 86-531-82169356; E-mail, [email protected]
Xian-Xi LIU: Tel,
86-531-82169436; Fax, 86-531-82169356; E-mail, [email protected]
Abstract Caveolin-1 is regulated by estrogen in vascular smooth muscle cells.
Raloxifene, a selective estrogen receptor modulator
that possibly has cardioprotective properties without an increased risk of cancer
or other side effects of estrogen, may be used in women with risk of coronary
artery disease. However, the relationship between raloxifene and caveolin-1 is
still unknown. Therefore, this study was designed to see whether raloxifene
regulates caveolin-1 expression and if so, whether such regulation is mediated
by estrogen receptor. Rat aortic smooth muscle cells were cultured in the
absence or presence of raloxifene (10–8 to 10–6 M) for 12 or 24 h. Both mRNA and protein levels of caveolin-1 were
increased significantly after 24 h
treatment with raloxifene. These increases were inhibited by estrogen
receptor antagonist ICI 182780 (10–5 M).
Results of this study suggest that raloxifene stimulates caveolin-1
transcription and translation through estrogen receptor mediated mechanisms.
Key words raloxifene; caveolin-1; estrogen receptor; vascular smooth muscle
cell
Caveolae, the flask-shaped vesicular invaginations of the plasma membrane,
are present in many cell types including vascular smooth muscle cells.
Caveolae have been implicated to be important for cellular functions such as
signaling, transport and proliferation [1-3]. The principal coat
proteins of caveolae are the caveolins. Thus far, three distinct mammalian
caveolin genes have been identified, and caveolin-1, a protein of 21–22 kDa, is so
far the best biochemical marker for caveolae [4–6].
Women experience initial manifestations of coronary artery disease
10 years later than men, suggesting that estrogen might play a cardioprotective
role. In vascular smooth muscle cells, estrogen stimulated the binding of ERa with caveolin-1
and augmented the production of caveolin-1 through a transcriptional mechanism
[7]. Mice lacking the caveolin-1 gene show impaired
endothelium-dependent relaxation, contractility and maintenance of myogenic
tone of the aorta [3]. Thus, estrogen-mediated upregulation of caveolin-1 might
be related to the improvement of vascular function [8].
Raloxifene belongs to a class of drugs recently described as
selective estrogen receptor modulators. It binds to the estrogen receptor and
shows tissue-specific effects such as estrogen agonist effects on bone and
lipids, and estrogen antagonist effects on the breast and uterus. Because the
side effects, especially the high risk of breast cancer, prohibit the use of
estrogen as a chronic prophylactic therapy for many postmenopausal women who
are at risk of cardiovascular disease, more tissue-specific agents, such as
raloxifene, may provide a good alternative. Recent findings suggested that
raloxifene also possesses beneficial effects on the cardiovascular system [9–12].
However, there are no reports demonstrating the relationship between
raloxifene and caveolin-1 in vascular smooth muscle cells. Hence, the present
study was designed to determine the effects of raloxifene treatment on
caveolin-1 expression in cultured rat aortic smooth muscle cells (RASMCs).
Materials and Methods
Chemicals
Raloxifene and 17b-estradiol were purchased from Sigma. ICI 182780 was bought from
Tocris. Anti-caveolin-1 antibody and anti-b-actin antibody were
purchased from Santa Cruz (Santa Cruz, USA). All other reagents and solvents
used in this study were of analytical grade.
Cell culture
RASMCs were isolated from the aorta of 8-week-old female Wistar rats
by enzymatic digestion, according to the method described previously [13]. The
cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented
with 10% fetal bovine serum (FBS), penicillin (100 U/ml) and streptomycin (100 mg/ml) at 37 ºC
in a humidified atmosphere of 95% air and 5% CO2.
Dextran-coated charcoal-stripped FBS (DCC-FBS) and phenol red-free DMEM were
used to avoid contaminations of steroids and estrogen receptor agonist.
Subcultured RASMCs (4–8 passages) were used in the following experiments.
RT-PCR analysis of caveolin-1 mRNA in RASMCs
RASMCs were seeded in 10 cm-culture dishes and grown to 80%–90% confluence.
The medium was then replaced with phenol red-free DMEM containing vehicle, 17b-estrodial (10–7 M), raloxifene (10–8 to 10–6 M), or in combination with ICI 182780 (10–5 M). Experiments were terminated after 12 h or 24 h of incubation
with the above agents. Cells were washed twice with phosphate-buffered saline
(PBS) and homogenized immediately in Trizol reagent. Total RNA was extracted
according to the manufacturer’s instructions. RT-PCR was performed using the
primers based on the rat caveolin-1 cDNA sequence. The sequences of primers for
caveolin-1 were: 5‘-GCATCCTCTTTCCTGCA-3‘ (sense), 5‘-TGGAATTAGCACGGCTGATG-3‘
(antisense). b-actin was used as a control for the quantity of the RNA. The
product of b-actin is a 275 bp fragment and the sequences of the primers were: 5‘-CTACAATGAGCTGCGTGTGGC-3‘
(sense) and 5‘-CAGGTCCAGACGCAGGATGGC-3‘ (antisense). The
amplification was performed with denaturation at 95 ºC for 30 s, annealing at
55 ºC for 30 s, and extension at 72 ºC for 60 s for 31 cycles with an initial denaturation
at 95 ºC for 5 min and a final extension of 5 min at 72 ºC. The PCR product was
fractionated by size on 2% agarose gels and visualized with ethidium bromide.
Western blot analysis of caveolin-1 protein in RASMCs
The confluent RASMCs were exposed to phenol red-free DMEM-containing
vehicle, 17b-estrodial (10–7 M),
raloxifene (10–8 to 10–6 M) or in combination with ICI 182780 (10–5 M) for 12 h or 24 h. Cells were then rinsed twice with PBS and lysed
in an ice-cold lyses buffer consisting of 20 mM Tris-HCl (pH 7.5), 150 mM
NaCl, 2 mM EDTA, 2 mM EGTA, 50 mM NaF, 1% Nonidet P-40, 1 mM
phenylmethylsulfonylfluoride, 1 mM Na3VO4 and 0.02 mM leupeptin. The resulting lysates were cleared by
centrifugation. The protein in the supernatant was quantified using Bradford
assay. Total protein (40 mg) from each sample was subjected to 10% SDS-PAGE for 1.5 h and
electroblotted onto nitrocellulose membranes for 3 h. The membrane was blocked
for 6 h at room temperature in TBS containing 5% skimmed milk powder, followed
by 2 h with the appropriate primary antibody (polyclonal rabbit
anti-caveolin-1 polyclonal antibodies, 1:500; rabbit anti-b-actin
polyclonal antibodies, 1:500). Afterwards, membranes were washed 4 times (15
min each time) in TBS and incubated with horseradish peroxidase labeled
anti-rabbit IgG secondary antibody for another 2 h. Finally, the immunoblots
were visualized using 3,3‘-diaminobenzidine-tetrachloride (DAB)
solution. The relative expression levels of caveolin-1 (caveolin-1/b-actin) were
measured using an imager in terms of the absorbance.
Statistical analysis
RT-PCR and Western blot experiments were carried out independently in
samples prepared from a minimum of four separate cultures. Data were presented
as mean±SEM. Analysis was performed with SPSS in Windows. Statistical
comparison was determined by analysis of variance. P<0.05 was
considered as being significantly different.
Results
Effects of raloxifene and 17b-estrodial on caveolin-1
expression
Compared with controls, the mRNA and protein expressions of
caveolin-1 were upregulated significantly by raloxifene (10–8 to 10–6 M), as well as 17b-estrodial (10–7 M) after 24 h treatment. No significant difference could be found
between 17b-estrodial and raloxifene, and no dose-dependent response effect of
raloxifene could be found (Figs. 1 and 2).
Time- and dose-dependent responses of raloxifene
In order to assess the effects of raloxifene on caveolin-1 fully,
further experiments were performed. As shown in Figs. 3 and 4,
the expression of caveolin-1 mRNA was increased after 24 h treatment of cells
with raloxifene. The increase in mRNA coincided with a statistically
significant increase in protein expression. Both caveolin-1 mRNA and protein
were observed to increase with the low dose of raloxifene (10–8 M) and not to increase further with high concentration of
raloxifene (10–6 M). Treatment with raloxifene
for 12 h did not significantly alter caveolin-1 mRNA and protein expressions (Figs.
3 and 4).
Influence of ICI 182780 on the effect of raloxifene
The pure estrogen receptor antagonist ICI 182780 (10–5 M) inhibited the increase of caveolin-1 mRNA and protein
expressions in response to raloxifene treatment for 24 h (Figs. 5 and 6).
Discussion
Results of the present study demonstrate for the first time the relationship
between raloxifene and caveolin-1 in vascular smooth muscle cells. Caveolin-1
mRNA and protein expressions increased in response to raloxifene treatment in
RASMCs. The pure estrogen receptor antagonist ICI 182780 inhibited the
increase, which suggests that raloxifene stimulates caveolin-1 transcription
and translation through estrogen receptor-mediated mechanisms. The lack of a
dose-dependent response effect of raloxifene treatment on caveolin-1 expression
might reflect saturation of the estrogen receptors with the lowest dose of
raloxifene. Caveolin-1 expression directly correlates with the caveolae number
[14,15]. Therefore, the results also suggest that raloxifene might influence
the number of caveolae present on the cell surface through regulation of
caveolin-1. Consistent with previous reports, the present study also showed
that estrogen upregulated caveolin-1 in vascular smooth muscle cells.
Though recent findings suggest that raloxifene might possess
cardioprotective properties such as reducing total and low-density lipoprotein
cholesterol, decreasing homocysteine levels and inhibiting vascular smooth
muscle cell proliferation [9–12], the exact role of raloxifene in the cardiovascular system has
remained unresolved until now. Recent studies in vivo and in vitro
dramatically show that caveolae and caveolin-1 play prominent roles in various
pathobiological conditions, especially those related to the cardiovascular
system. Caveolin-1 contains a cytosolic N-terminal juxtamembrane domain (scaffolding
domain), which binds to signaling molecules and inhibits their usual activation
after growth factor ligation of receptors [16,17]. Such examples include
receptor tyrosine kinases, serpentine receptors and regulated enzymes. The
interactions of caveolin-1 with these signaling molecules have important
consequences for cellular functions such as proliferation. In the development
of atherosclerosis, the proliferation of smooth muscle cells is a crucial
pathophysiological process. Recent findings reported that caveolin-1 could
negatively regulate the proliferative activity of vascular smooth muscle cells
and inhibit neointimal hyperplasia [18,19]. Caveolin-1 is also thought to play
an important role in the regulation of cellular cholesterol homeostasis, a
process that needs to be properly controlled in order to limit and prevent
cholesterol accumulation and eventually atherosclerosis. For example, Batetta et
al. [20]
found that compared with adjacent serial sections of
the same artery, atherosclerotic segments manifested higher levels of
cholesterol esters, ACAT (acyl-coA:cholesterol acyltransferase) and multidrug
resistance 1(MDR) mRNA and lower levels of caveolin-1 mRNA. Therefore,
upregulation of caveolin-1 by raloxifene in vascular smooth muscle cells might
contribute to protect the cardiovascular system against atherogenesis.
The present study also demonstrated that the upregulation effect of
raloxifene on caveolin-1 expression was blocked by ICI 182780. Two types of
estrogen receptors, estrogen receptors a and b were found in
vascular smooth muscle cells, and emerging evidence shows that there is a
plasma membrane estrogen receptor that could associate with and regulate the
production of caveolin [7]. It is unknown whether this blockade is mediated by
estrogen receptor a or b, and it is also unclear whether this blockade is related to the
nuclear estrogen receptor or membrane estrogen receptor. A further study is
needed to clarify the molecular mechanism by which raloxifene upregulates
caveolin-1 mRNA and protein expressions.
In summary, raloxifene could influence the progression of coronary
disease and the incidence of cardiovascular events through several mechanisms.
Results of the present study demonstrate that raloxifene increases gene
transcription and translation of caveolin-1 in vascular smooth muscle cells,
providing a new pathway through which raloxifene affects vascular functions.
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