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https://www.abbs.info ISSN 0582-9879 |
Mouse
Restin Inhibits Bovine Aortic Endothelial Cell Proliferation and Causes Cell
Apoptosis
XU
Ren, XIN Li, FAN Yi1, MENG Hai-Rong, LI Zai-Ping, GAN Ren-Bao*
(
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Science,
Shanghai
200031, China; 1Shanghai University of Traditional Chinese
Medicine, Shanghai 200032, China )
Abstract Restin,
a homologous protein of endostatin, was found by Ramchandran et al. It was the C-terminal fragment of type
XV collagen. To analysis the
inhibition activity of mouse restin on the proliferation of endothelial
cells, the cDNA of restin was
amplified from the total RNA of the mouse muscle and cloned into the
prokaryotic expression plasmid pQE32.
The recombinant protein was expressed in inclusion body with a yield
about 60%―70% of total protein. After refolding, the purified recombinant protein
specifically inhibits bovine aortic endothelial (BAE) cell proliferation
stimulated by basic fibroblast growth factor (bFGF) in a dose-dependent
manner, but the activity of restin
was weaker than that of endostatin.
Treatment of BAE cell with recombinant restin caused G1
arrest and apoptosis in BAE cells.
Key
words restin; endostatin; collagen XV;
angiogenesis inhibitor;
apoptosis
O’Reilly
et al[1] isolated endostatin, an angiogenesis inhibitor isolated from a murine
hemangioendothelioma cell line[1]. The N-terminal sequence of endostatin revealed identity to
the C-terminal portion of collagen XVIII.
It consists of 185 amino acids.
Endostatin specifically inhibits endothelial proliferation and potent
inhibits angiogenesis and tumor growth.
Treatment of cow pulmonary artery endothelial cell with endostatin
causes apoptosis. Systemic therapy
with non-refolded precipitated protein expressed in E.coli caused
regression of primary tumors[1―4].
A
significantly homologous protein found by Ramchandran et al was the
C-terminus 184 amino acids of mouse collagen XV alpha 1 chain. Type XV collagen is composed of 1 363
amino acids. It contains a highly
interrupted collagenous (COL) region of 577 residues, and noncollagenous (NC) amino- and carboxyl-terminal domain
of 530 and 256 residues[5].
Type XV collagen presented in many tissues as evidenced by strong
association with vascular,
neural, mesenchymal and
some epithelial basement membrane zones[6]. Ramchandran et al[７]
found that the recombinant C-terminus of human collagen XV (restin) could
inhibit the migration of endothelial cells in vitro but had no effect on
the proliferation of these cells.
In our experiments, the
result showed that recombinant mouse restin could inhibit the proliferation of
endothelial cells in vivo.
Treatment of BAE cells with recombinant restin caused G0/G1
arrest and cell apoptosis.
The
cDNA of C-terminus of collagen XV alpha 1 chain was amplified from total RNA of
mouse muscle and was cloned into the expression plasmid pQE32. After induction with IPTG, the recombinant protein was expressed
in inclusion body and accounted for 60%―70%
of the total E.coli protein.
The recombinant protein was purified and refolded. It inhibited BAE cell proliferation
stimulated by bFGF as a dose-dependent manner. Flow cytometry and annexin V-FITC binding assay demonstrated
that treatment of BAE cell with recombinant protein caused the changes of cell
cycles and cell apoptosis.
1 Materials and Methods
1.1
Reagents and materials
DMEM
and Trypsin/EDTA were purchased from Gibco BRL(Rockville, MD). Fetal calf serum was from Hyclone (Logen UH). Mouse was from Shanghai Animal
Center. Endostatin was prepared in
our lab. BAE cells were isolated
as previously reported from bovine aortic[8].
1.2
RT-PCR
Mouse
fresh muscle was homogenized in liquid nitrogen. Total RNA was isolated using
Trizol reagent (Gibco BRL). Total
RNA was used as the template for cDNA synthesis using SuperscriptTM
RNase H– transcriptase (Gibco BRL) according to manufacturer’s
instructions. PCR was performed with Ex-Taq DNA polymerase (TaKaRa)
according to manufacturer’s instructions. The synthetic oligo-nucleotides were
obtained from Shanghai Sangon Co. Ltd. (Shanghai, China). The primers used were
as follows: RT primer: 5′-TTATTACTTCCTAG-TGTCTGTCATGAAAC-3′,
sense primer: 5′-ATT-TTAAGTGCCAACTATGAGAGGCCT-3′,antisense
primer: 5′-TTATTACTTCCTAGTGTCTGTCATG-AAAC-3′,
two stop codons were included in the antisense primer. PCR product of 560 bp was amplified
with this primer sets. Reaction
was incubated in PE480 thermal cycler (Perkin-Elmers, NJ) for 35 cycles: denaturation 30 s, 94 ℃; annealing 30 s, 55 ℃; extension 30 s, 72 ℃. PCR product was run on 1% agarose gel
in TBE (10 mmol/L Tris-borate, 1
mmol/L EDTA, pH 8.0), and visualized by ethidium bromide
staining.
1.3
Plasmid construction
The
amplified cDNA fragment was ligated into Escherichia coli
expression vector pQE32 (Qiagen),
resulting in the construction of an expression plasmid pQEXV. The restin containing vector was
sequenced.
1.4
Purification and refolding of recombinant restin
pQEXV
was transformed into E.coli M15 (Qiagen) and mouse restin expression was
induced by 1 mmol/L IPTG. Cells
were harvested by centrifugation for 10 min at 4 000 g. Cells were resuspended in 20 mmol/L
Tris-HCl, pH 8.0, 50 mmol/L KCl, 0.5 mmol/L EDTA, 5 mmol/L DTT and lysozyme was added to
the final concentration of 0.5 g/L.
Cells were incubated at 4 ℃
for 30 min, then were disrupted by
sonic homogenizer for 10 s for ten times with 30 s interval each time. After centrifugation at 4 ℃，
12 000 g for 30 min, the pellet was collected and resuspended in 8 mol/L
urea, 0.1 mol/L NaH2PO4, 10 mmol/L Tris-HCl, pH 8.0. Centrifuged again as
before, the supernatant was loaded
on a Ni2+-nitrilotriacetic acid-agarose column (Qiagen). The recombinant protein was eluted from
the column according to manufacturer’s instructions. To achieve refolding,
the purified protein was adjusted to pH 8.0 and DTT was added to the
final concentration of 0.01 mol/L.
Following incubation at room temperature for 2 h, the solution was added to refolding
buffer (0.1 mol/L Tris-HCl, pH
8.0, 0.5 mol/L arginine, 5 mmol/L EDTA, 1 mmol/L GSSG, 5 mmol/L GSH) with the ratio of 1∶200[9]. After 24 h incubation at room
temperature, the renatured protein
was dialyzed against PB buffer (10 mmol/L Na2HPO4, 10 mmol/L NaH2PO4, pH 7.2) for 24―48
h and lyophilized.
1.5
Bovine aortic endothelial cell proliferation assay
Bovine
aortic endothelial cells were isolated as previously described[8]
and maintained in DMEM supplemented with 10% heat-inactivated FCS and
antibiotics. Monolayer of BAE
cells growing in 60 mm dish was dispersed in 0.05% trypsin solution. Cells were resuspended with DMEM
containing 2% FCS. Approximately 3
000 cells in 200 ml
were added in triplicate to each well of 96-well tissue culture plates and
incubated at 37 ℃
(in 5% CO2). Cells
adhered to the plate in about 2―3
h. The medium was replaced with
200 ml
of fresh DMEM containing 2% FCS, 5
mg/L
bFGF, and samples of recombinant
restin or endostatin were added to each well. After 72 h incubation,
10 ml
MTT (100 g/L) was added to each well and incubated for another 4 h at 37 ℃, 5% CO2. 180 μl
medium was pipetted out from each well and 50 μl
DMSO was added, vortex gently to
dissolve the pellet[9, 10]. The absorbency A570, which correlates to the number of
cells, was measured with
microplate reader (Model 450,
Bio-Rad).
1.6
Cell cycle analysis
All
the procedures were followed as previously reported[11]. Briefly, BAE cells were maintained in DMEM supplemented with 10% FCS
till to 60%―70%
confluence. The medium was changed
with DMEM supplemented with 2% FCS containing 2 mg/L recombinant restin and
bFGF was added to the final concentration of 5 mg/L. After 24 h incubation, cells were trypsinized and washed
gently with PBS, and then were
fixed with 70% ice-cold ethanol for 30 min. Cells were collected by centrifugation. 200 ml
1 g/L RNase was added and incubated at 37 ℃
for 30 min, followed by staining
with propidium iodide at 5 mg/L.
Cells were assessed by FACStar plus flow cytometer (Beckton-Dickinson)
and the results were analyzed with CellQuest software[11].
1.7
Annexin V-FITC binding assay
Annexin V-FITC
(Clontech, Palo Alto, CA) binding assay was performed
according to manufacturer’s instruction.
2.5×105
cells were plated onto a 60 mm dish in DMEM containing 2% FCS. After 24 h incubation at 37 ℃, 5% CO2, the medium was changed with DMEM
supplement with 2% FCS, 5 mg/L
bFGF and 2 mg/L recombinant restin.
After 24 h incubation,
cells were trypsinized and were washed in PBS and resuspended in binding
buffer (10 mmol/L HEPES/NaOH, pH
7.4, 140 mmol/L NaCl, 2.5 mmol/L CaCl2). Annexin V-FITC was added to a final
concentration of 100 mg/L, and the cells were incubated in the
dark for 10 min. For each
sample, minimums of 10 000 cells
were counted. Data analysis was
performed with standard Cell Quest software[12, 13].
2
Results
2.1
Cloning the restin gene in expression plasmids
The mouse restin gene
was amplified by RT-PCR using total RNA from mouse muscle with the primers we
designed. The cDNA encodes a
portion of 184 amino acids corresponding to the amino acid positions 1 132 to 1
315 of the collagen XV. The
amplified cDNA fragment was cloned into the E.coli expression vector
pQE32. DNA sequence analysis
indicated that the desired plasmid had been obtained.
2.2
Purification and characterization of recombinant mouse restin
Recombinant
protein mouse restin plus six histidine was expressed in E.coli and
purified using Ni2+-nitrilotriacetic acid-agarose column and was
refolded in vitro (Fig.1).
Under reducing condition,
recombinant restin migrated in SDS-PAGE with molecular mass of about 22
kD, corresponding to the predicted
molecular mass.
Fig.1 Recombinant
production of mouse restin plus six histidine in E.coli
15% SDS-PAGE Coomassie blue staning; 1,
moleculor weight marker; 2, uninduced cells; 3, induced cells; 4, soluble
protein; 5, insoluble protein; 6, purified protein.
2.3
Inhibition of BAE cell proliferation with recombinant restin and mouse
endostatin
His-restin and
endostatin were assayed for their inhibitory activities on bovine aortic
endothelial cell growth stimulated by bFGF. As shown in Fig.2,
both endostatin and restin inhibited BAE cell proliferation in a
dose-dependent manner, but the
inhibitory activity of restin was weaker than that of endostatin. While both restin and endostatin has no
inhibitory activities on fibroblast cell line Balb/c3T3 and hepatoma cell line
7404 (data not shown), which
suggested that their inhibitory activity was specific to endothelial cell.
Fig.2 Recombiant mouse restin and endostatin
inhibit the proliferation of BAE cells stimulated by bFGF (n=3)
2.4
Cell cycle analysis
As shown in
Fig.3, BAE cells were treated with
2 mg/L restin overnight in 0.5% FCS. The cell cycle assay demonstrated that
50.9% of cells wasblocked in the G0/G1 after a 24 h
treatment with restin, compared with 29.8% of control. The percentage of S
phase cell was 31.2%, compared with 46.8% of control. These results may explain
partly the anti-proliferation effect of restin on BAE cell.
Fig.3 Flow cytomery of BAE cell treatment
with restin
BAE cell monolayers were exposed 24 h to
restin in DMEM supplement with 2% FCS, and cells were assessed by FACStar plus
flow cytometer (Decton-Dickinson). (A) control without restin. (B) cells
treated with restin (2 mg/L).
2.5
Cell apoptosis detection
Annexin V, a calcium-dependent
phospholipid-binding protein with a high affinity to phosphatidylserine
(PS), was used to detect the early
stage apoptosis[13]. As
shown in Fig.4, treatment with 2
mg/L recombinant restin or endostatin in 2% FCS caused BAE cell apoptosis. At the same concentration, the apoptosis-inducing activity of
endostatin was greater than that of restin, corresponding to the result of proliferation assay.
Fig.4 Annexin V-FITC binding assay
BAE cell were treated for 24 h with 2
mg/L restin (2) or 2 mg/L endostatin (3) or an equal volume of PBS(1). Detached
cells and attached cells were collected, and phosphatidylserine externalization
was mesured by labeling with FITC-labeled annexin V. The righword shift on the
x axis of the restin and endostatin peaks indicates increased annexin V-FITC
binding of apoptotic cells.
3
Discussion
Angiogenesis is
required for tumors to grow beyond a few millimeters in size[14]. Numerous studies have shown that both
primary tumor growth and metastasis is angiogenesis dependent[1]. Advantages of anti-angiogenic therapy
include ease of access of drugs to the endothelial cell compartment and
lessening the chance of drug resistance.
A number of angiogenesis inhibitor has been identified. Such as angiostatin and endostatin are
fragments of proteins. Endostatin, a 20 kD C-terminal fragment of collagen
XVIII, is a specific inhibitor of
endothelial cell proliferation and migration.
Restin, a homology protein of endostatin, is the C-terminus of the collagen
XV. Reports by Ramchandran et al[7]
showed that the human restin could inhibited the migration of endothelial cells
in vitro and angiogenesis in mouse model, but didn’t inhibit the proliferation of endothelial
cells. In this report, we amplified the gene of 3′terminus
of collagen XV from mouse muscle total RNA. The gene was cloned into pQE32 plasmid. The recombinant protein was expressed, purified and refolded. The recombinant protein inhibited BAE
cell growth as a dose-dependent manner.
Endostatin was reported to induce endothelial cell apoptosis[15]. We here demonstrated that mouse restin
could induce BAE cell apoptosis,
too. Cell cycle analysis of
BAE cells cultivated in medium with mouse restin showed a cell arrest mainly in
the G0/G1 phase.
These results correlate with the BAE cell proliferation assay described
above. There are 20 amino acids
different between mouse and human restin (Fig.5), and these difference may be the reason why the two proteins
have the different biological activity.
Fig.5 The sequence alignment of mouse and
human endostain, restin
The
same amino acids residues are marked with capital letter.
The effects of restin
and endostatin on the proliferation of BAE cells were compared. The result demonstrated that the
anti-proliferation effect of recombinant restin was weaker than that of
endostatin. Cell cycle analysis
and apoptosis detection also showed the G1 arrest and cell apoptosis
caused by endostatin was greater than that caused by restin. The sequence alignment showed restin
has 64% homology with endostatin (Fig.5).
This may be the reason why they have the similar activity. Recent study demonstrated that
tropomyosin was a potential modulator of the antitumor activity of endostatin. They showed that recombinant human
endostatin binds tropomyosin in vitro and to tropomyosin-associated
microfilaments in a viriety of endothelial cell types[16]. Since restin is high homology with
endostatin, it may inhibit cell
proliferation and induce cell apoptosis by binding to tropomyosin.
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Received: September 7, 2001 Accepted: October 22, 2001
*Corresponding author: Tel, 86-21-64374430-5325; Fax, 86-21-64338537; e-mail, [email protected]