From the Department of Pathology, University of
Washington, Seattle, Washington 98195 and the § Division of
Cardiology, Mitsui Memorial Hospital, Tokyo 101-8643, Japan
Received for publication, September 18, 2000, and in revised form, November 10, 2000
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ABSTRACT |
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Serum depletion induces cell death. Whereas serum
contains growth factors and adhesion molecules that are important for
survival, serum is also likely to have antiapoptotic factor(s). We show here that the plasma proteinase inhibitors Serum depletion has been demonstrated to induce apoptosis (1).
Integrin-mediated adhesion to extracellular matrix is required for
growth and survival of many cell types. This is due in part to the fact
that adhesion to extracellular matrix is required for progression of
cells through the cell cycle by regulating cyclin D1 and cyclin
E-Cdk2 (2). Disruption of adhesion arrests cells in the
G1 phase and causes apoptosis (3-8). Signaling mediated by
It is sometimes difficult to keep smooth muscle cells in culture
without serum despite the presence of extracellular matrix proteins and
growth factors. This observation suggests that serum contains
unidentified survival factors.
In a previous study, we showed that Induction of Cell Death--
Cultured human vascular smooth
muscle cells (HNB18E6E7) were originally derived from the aorta of a
newborn infant (2-day-old) autopsy (11, 12). The cells were detached by
brief exposure to 0.05% trypsin, 0.02% EDTA. Trypsin was inactivated
by excess amounts of soybean trypsin inhibitor (Sigma). After washing
with PBS, the cells were resuspended in serum-free DMEM containing 0.2% bovine serum albumin, at a concentration of 50,000 cells/50 µl.
A 96-well culture plate was previously coated with either fibronectin
(10 µg/ml) or vitronectin (10 µg/ml) at room temperature for 1 h. The cell suspensions (50 µl) were plated on the 96-well culture
plate. 50 µl of each sample were overlaid. Final concentration of the
samples was 5% calf serum, PDGF-BB (20 ng/ml), Western Blot Analysis--
After observing cell morphology, 25 µl of 25% SDS was added to the wells and shaken for 1 h. The
samples were run on an 8% SDS-polyacrylamide gel electrophoresis, and
proteins were transferred to polyvinylidene difluoride membrane
(Bio-Rad). The membrane was blotted with polyclonal anti-fibronectin
antibody (R790), a kind gift provided by Dr. William G. Carter (Fred
Hutchinson Cancer Research Institute, Seattle, WA), at 1:10,000
dilution, and the antigen was visualized with Renaissance Western blot
chemiluminescence reagent (PerkinElmer Life Sciences).
Trypan Blue Exclusion Assay--
After collecting floating
cells, attached cells were exposed to 0.05% trypsin, 0.02% EDTA.
Trypsin was inactivated by soybean trypsin inhibitor (Sigma). All the
attached and detached cell populations were combined to determine the
proportion of dead cells. Trypan blue (Life Technologies, Inc.) was
mixed with cells (1:1), and trypan blue exclusion by living cells was
scored using phase contrast microscopy.
TUNEL Staining--
TUNEL staining was performed using the
in situ Cell Death Detection Kit, Fluorescein (Roche
Molecular Biochemicals cat. No. 1 684 795). The nuclei were
counterstained with Hoechst at 5 µg/ml.
Inactivation of Effect of z-VAD--
A 96-well culture plate was previously
coated with fibronectin. Cell suspension of 50,000 cells/94.5 µl were
plated. Each sample was overlaid; 0.5 µl of 20 mM z-VAD
in Me2SO plus 5 µl of PBS, 0.5 µl of
Me2SO plus 5 µl of 0.125 mg/ml Assay for Caspase Activity--
Activity of caspase was
determined using fluorescent caspase-specific substrate PhiPhiLux-G1D2
(Oncoimmunin) of which the main peptide sequence is GDEVDGI. Human
vascular smooth muscle cells were cultured on a plate previously coated
with fibronectin for 24 h with Statistics--
All experiments were repeated three times.
Analysis of variance test was performed using SAS software version 6 in
order to compare mean and variance.
Human vascular smooth muscle cells (HNB18E6E7) were plated in DMEM
medium containing either calf serum, PDGF-BB, 1-proteinase inhibitor,
1-antichymotrypsin, and
2-macroglobulin function as critical antiapoptotic factors for human vascular smooth muscle cells. Cell
survival was assured when serum-free medium was supplemented with any
one or all of the above serine proteinase inhibitors. In contrast, the
cells were sensitive to apoptosis when cultured in medium containing
serum from which the proteinase inhibitors were removed. The
antiapoptotic effect conferred by the proteinase inhibitors was
proportional to proteinase inhibitory activity. Without proteinase
inhibitors, the extracellular matrix was degraded, and cells could not
attach to the matrix. Cell survival was dependent on the intact
extracellular matrix. In the presence of the caspase inhibitor
z-VAD, the cells detached but did not die. The activity of caspases was
elevated without proteinase inhibitors; in contrast, caspases were not
activated when medium was supplemented with one of the proteinase
inhibitors. In conclusion, the plasma proteinase inhibitors prevent
degradation of extracellular matrix by proteinases derived from cells.
Presumably an intact cell-matrix interaction inhibits caspase
activation and supports cell survival.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3-integrin stimulates NF-
B, which results in cell survival (9).
Therefore, extracellular matrix proteins are required in addition to
growth factors for cell survival.
1-proteinase inhibitor
(
1PI),1
1-antichymotrypsin (
1ACT), and
2-macroglobulin (
2M) are
necessary for cell spreading and adhesion in fibrin gels (10). These
proteinase inhibitors protect degradation of extracellular matrix
proteins by cell-derived proteinases. Thus, the plasma proteinase
inhibitors are essential to ensure appropriate cell-matrix
interactions. Analysis of the cells that detach in the absence of
proteinase inhibitors showed a similarity to apoptotic cells induced by
serum depletion. Based on these results, we hypothesized that the
proteinase inhibitors function as critical survival factors in vascular
smooth muscle cells.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1PI (0.125 mg/ml),
1ACT (0.125 mg/ml), and
2M (0.125 mg/ml) in DMEM with 0.2%
bovine serum albumin. The plates were incubated at 37 °C with 5%
CO2 for 24 h. Attached cells were counted using phase contrast microscopy. Fibronectin, vitronectin,
1PI,
1ACT, and
2M were purchased from CalBiochem. PDGF-BB was from Life
Technologies, Inc. The experiments were repeated three times.
1PI and
2M--
1PI was inactivated
according to Johnson and Travis (13); 0.1 mg of human
1PI
(CalBiochem) in 40 µl of 0.1 M Tris buffer, pH 8.8 was
mixed with 10 µl of 80 mM N-chlorosuccinimide
(Sigma) and incubated overnight. The excess of the reagent was removed by dialysis. The remaining antitrypsin activity was less than 1 × 10
4 of the original activity.
2M (CalBiochem) was
inactivated by treating with 0.2 M methylamine (Sigma) in
50 mM Tris, pH 8.0 for 6 h at room temperature. After
dialysis, the remaining activity was less than 1 × 10
4.
2M in PBS, or 0.5 µl
of Me2SO plus 5 µl of PBS. Final concentration of z-VAD was 0.1 mM and that of
2M was 6.25 µg/ml. Cell
morphology and trypan blue exclusion were performed as noted above.
2M at 1.7 × 10
7 M, without
2M, with z-VAD at 1 × 10
4 M or with staurosporin at 1 × 10
6 M. Cells were collected, then washed in
PBS and incubated at 37 °C for 1 h with 50 µl of 10 µM substrate solution. Flow cytometry analysis was
performed at
ex = 505 nm and
em = 530 nm.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1PI,
1ACT, or
2M
on 96-well culture plate previously coated with either fibronectin or
vitronectin. PDGF-BB induces cell division of HNB18E6E7 cells as shown
by increased thymidine incorporation (data not shown). Attached cells
were counted using a phase contrast microscope. At 1 h, cell
attachment on the surface was similar. However, at 24 h, the
numbers of attached cells were significantly different (Fig.
1A). Fig. 1B shows
photographs at 24 h, when cells had detached and looked round upon
serum depletion or in the presence of PDGF-BB. In contrast; with serum,
1PI,
1ACT, or
2M; cells remained attached and spread. A number
of cells with calf serum was about 10% more than
1PI,
1ACT, or
2M, probably because the growth factors in serum
supported proliferation.
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Fig. 1.
Cell attachment on fibronectin.
A, vascular smooth muscle cells were cultured on a plastic
surface previously coated with fibronectin. Culture medium was DMEM
with 0.2% bovine serum albumin, pH 7.4, containing either 5% calf
serum (CS), no serum (N), platelet-derived growth
factor (PDGF) at 20 ng/ml, 1-proteinase inhibitor
(PI),
1-antichymotrypsin (ACT), and
2-macroglobulin (M) at 0.125 mg/ml. Attached cell numbers
were counted using a phase contrast microscope at 1 h and 24 h. N and PDGF were significantly lower than the other four groups,
p < 0.001. B, photographs were taken at
24 h with a phase contrast microscope. Previous coat on plastic
surface was either fibronectin (FN) or vitronectin
(VN) (original magnification, × 100). C, Western
blot analysis for fibronectin. After observation of cell morphology as
shown in B, the coated fibronectin was solubilized with 5%
SDS. Each sample was electrophoresed on 8% SDS-polyacrylamide gels,
transferred to a polyvinylidene difluoride membrane, and blotted with a
polyclonal antibody against fibronectin.
To determine whether the endogenous cellular proteinases
disrupted the cell matrix, we examined the integrity of fibronectin. Specifically, fibronectin was extracted by 5% SDS, and then Western blot analysis was performed. Fibronectin was intact with serum, 1PI,
1ACT, or
2M; however, it was degraded with PDGF or no serum (Fig.
1C). Therefore, the morphology of the cells depended on the
integrity of adhesion molecules.
To determine how many cells were dead, we collected all the detached
and attached cells and then counted the total number of dead cells by
trypan blue exclusion test (Fig.
2A). Dead cell numbers were
significantly higher with no serum or PDGF alone than with serum or
proteinase inhibitors. Next, we confirmed apoptosis using TUNEL
staining although TUNEL detects apoptosis only of attached cells (Fig.
2B). TUNEL positivity was significantly higher with no serum
or PDGF than other groups. These data suggest that proteinase
inhibitors prevent degradation of matrix, which supports cell
attachment and may result in cell survival.
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Next, we quantified the survival effect of the proteinase inhibitors.
1PI,
1ACT, and
2M induce cell survival in a
dose-dependent manner by the trypan blue exclusion test
(Fig. 3A). In this assay, 50%
survival was achieved at 4.4 × 10
8 M in
2M, at 3.3 × 10
7 M in
1PI, and at
4.2 × 10
7 M in
1ACT. Thus,
2M has
a 7.6 times higher ability than
1PI and 9.6 times higher than
1ACT. We next removed both
1PI and
2M from human serum by
immunoprecipitation. We did not have an appropriate antibody against
1ACT and thus could not deplete it. Ninety-five percent of the
proteinase inhibitory activity was lost by the simultaneous removal of
both proteins. The remaining 5% activity was probably due to the
presence of
1ACT. The immunoprecipitated serum lost ~95%
of survival activity, assessed by trypan blue (Fig. 3B).
Furthermore, we tried an admixture of three proteins at normal serum
level. Survival rate with the admixture was not statistically different
from that with serum in this assay (Fig. 3B).
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Chemical inactivation was performed on 1PI and
2M.
1PI was
inactivated with N-chlorosuccinimide.
2M was inactivated with methylamine. Both
1PI and
2M lost their survival effect (Fig. 3C). These results indicate that the ability of cell
survival is attributable to proteinase inhibitory activity.
We compared the proteinase inhibitors with a caspase inhibitor as
survival factors. Cells attached on a plastic surface previously coated
with fibronectin at 24 h with 2M (Fig.
4A); however, cells detached
with no serum (Fig. 4B) or with z-VAD (Fig. 4C).
The level of attached cells was significantly higher with
2M than other groups (Fig. 4D). In contrast, with z-VAD, the cells
are alive (assessed by trypan blue exclusion) although many cells have
detached (Fig. 4E). z-VAD had no effect on attachment although detached
cells survived with z-VAD.
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Next, we determined caspase activity using caspase-specific fluorescent
substrate, PhiPhiLux-G1D2. With 2M, caspase activity was as low as
with z-VAD (Fig. 5). However, without
2M, caspase activity was significantly high, similar to that with
staurosporin. This suggests that the proteinase inhibitor not only
supports attachment but also prevents activation of caspases.
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The data suggest a novel mechanism for cell survival induced by plasma
proteinase inhibitors. Cell survival is dependent on the ability of
proteinase inhibitor to protect adhesion matrix. If the proteinase
inhibitors are not present, the matrix is degraded by proteinases, and
the cells cannot maintain attachment.
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DISCUSSION |
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It is well documented that extracellular matrix and integrin
interactions generate intracellular signals that lead to caspase activation (14-16). However, combinations of adhesion molecules and growth factors alone are often insufficient to keep cells in
culture alive without serum. Serum therefore, has been assumed to
include unidentified survival factor(s). We have demonstrated that the
critical antiapoptotic factors in serum are 1PI,
1ACT, and
2M.
We also show that the mechanism of survival involves proteinase
inhibitors, protecting the extracellular matrix from being degraded by
cell-derived proteinase(s).
Cell migration is required during development, in tissue repair and in wound healing. Degradation of the extracellular matrix is a prerequisite for cell migration into the three-dimensional matrix. As part of this homeostatic process, cells produce proteinases, including matrix metalloproteinases (17) and/or serine proteinases. Careful regulation of proteolytic activity is required, as too much degradation will induce detachment that may result in cell death. In many cases, matrix proteinase activity is immediately neutralized. In addition to plasma proteinase inhibitors, many proteinase inhibitors such as tissue inhibitor of matrix metalloproteinases (TIMPS) have activity against specific cell-derived proteinases (18). Because a broad spectrum of proteases have apoptotic activity, it is reasonable to assume that many proteinase inhibitors are potentially antiapoptotic factors, acting via a mechanism similar to what we have shown here.
There is some controversy over whether TIMPs are proapoptotic or antiapoptotic. TIMP-3 induces apoptosis (19-22) via a death domain located within the N terminus (23). Alexander et al. (24) showed that overexpression of TIMP-1 prevents apoptosis of epithelial cells from stromelysin-1 transgenic mice. Several other studies have shown antiapoptotic effects of TIMP-1 (25, 26) and TIMP-2 (27). The role of proteinases is not limited to matrix degradation in vivo. For example, extracellular proteinases can directly modify intracellular signals via proteinase-activated receptors (28). Thus, the interaction between proteinases and proteinase inhibitors may influence a cells response to its environment in several ways.
Compared with the abundance of albumin and globulin, proteinase
inhibitors are the third most prevalent group of plasma proteins. In
our assay, neither albumin nor globulin demonstrated any antiapoptotic effect whereas 2M, a very nonspecific protease inhibitor, was very
cytoprotective.
2M is an ancient protein that is found in all
vertebrates and in non-vertebrates such as eels and crabs (29). There
is speculation that
2M is a carrier of transforming growth
factor-
and other cytokines (30) or that it functions as an agonist
of
2M receptors (31). No
2M genetic deficiency has been found in
our species despite extensive screening of human plasma (32).
Furthermore,
2M knock-out mice have no phenotype (33).
1PI is a
universal inhibitor of serine proteinases, which when diminished or
mutated causes pulmonary emphysema and liver fibrosis. Physiologically,
1PI is considered to be an elastase inhibitor (32).
1ACT is a
chymotrypsin type serine proteinase inhibitor.
1ACT is recognized as
an acute phase protein. Serum concentrations of
1ACT increase
rapidly and dramatically after a variety of events including surgery,
burn injury, inflammatory bowel disease, and some types of cancer (32).
This probably indicates that an increase in
1ACT supports the
survival of resident cells in areas of inflammation. Although the
actual roles of the plasma proteinase inhibitors are not completely
clarified, our data show a novel biological activity of these
proteinase inhibitors.
One obvious question is what proteinase(s) does the smooth muscle cell produce? Based on the specificity of proteinase inhibitors, chymotrypsin type serine proteinases are candidates. We observed that this cell line produced chymotryptic activity when co-cultured with a chymogenic substrate (data not shown). Additional experiments using degenerate PCR primers and screening of a large gene expression array also identified a trypsin-like protease (manuscript in preparation).
In conclusion, we have shown a novel anti-apoptotic activity of plasma
proteinase inhibitors. Because of the high concentration of 1PI and
2M, the proteinase inhibitors may function as the major
antiapoptotic proteins present in serum. The proteinase inhibitors
prevent extracellular matrix from degradation by cell-derived proteinase(s). This novel activity may suggest that the proteinase inhibitors could play a role in vascular disease such as
atherosclerosis or aortic aneurysm.
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ACKNOWLEDGEMENT |
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This study was supported by NIH HL-03174, ROI HL-61860 and Mitsui Life Social Welfare Foundation.
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FOOTNOTES |
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* This study was supported by National Institutes of Health Grants HL-03174 and ROI HL-61860 and the Mitsui Life Social Welfare Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Mitsui Memorial Hospital, 1, Kanda-Izumi-cho, Chiyoda-ku, Tokyo 101-8643, Japan. Fax: 81-3-5687-9765; E-mail: ikari-tky@umin.ac.jp.
Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M008503200
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ABBREVIATIONS |
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The abbreviations used are:
1PI,
1-proteinase inhibitor;
1ACT,
1-antichymotrypsin;
2M,
2-macroglobulin;
PDGF, platelet-derived growth factor;
PBS, phosphate-buffered saline;
DMEM, Dulbecco's modified Eagle's medium;
z-VAD, benzyloxycarbonyl-VAD;
TUNEL, terminal
deoxynucleotidyltransferase (TdT)-mediated dUTP nick-end
labeling.
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