(Received for publication, May 2, 1995; and in revised form, August 31, 1995)
From the
Plasminogen activator inhibitor type 2 (PAI-2) is a serine
proteinase inhibitor or serpin that is a major product of macrophages
in response to endotoxin and inflammatory cytokines. We have explored
the role of PAI-2 in apoptotic cell death initiated by tumor necrosis
factor (TNF). HeLa cells stably transfected with PAI-2 cDNA were
protected from TNF-induced apoptosis, whereas cells transfected with
antisense PAI-2 cDNA, a control gene, or the plasmid vector alone
remained susceptible. The level of PAI-2 expressed by different HeLa
cell clones was inversely correlated with their sensitivity to TNF.
Loss of TNF sensitivity was not a result of loss of TNF receptor
binding. In contrast, PAI-2 expression did not confer protection
against apoptosis induced by ultraviolet or ionizing radiation. The
serine proteinase urokinase-type plasminogen activator was not
demonstrated to be the target of PAI-2 action. The P
-Arg
amino acid residue of PAI-2 was determined to be required for
protection, because cells expressing PAI-2 with an Ala in this position
were not protected from TNF-mediated cell death. The results suggest
that intracellular PAI-2 might be an important factor in regulating
cell death in TNF-mediated inflammatory processes through inhibition of
a proteinase involved in TNF-induced apoptosis.
Programmed cell death is conserved throughout evolution as a
strategy employed by multicellular organisms to regulate a wide range
of physiological processes including embryo development, tissue
remodelling, immune system development and cellular responses to
infection, and tumorigenesis. Cell death can be triggered by a variety
of stimuli that can ultimately lead to characteristic changes in the
cell, frequently resulting in chromatin fragmentation and condensation,
membrane blebbing, and collapse of the nucleus, a process called
apoptosis(1, 2) . Within the immune system, tumor
necrosis factor (TNF) (
)is an important effector of
programmed cell death, playing a role in immune defense against viral,
bacterial, and parasitic infections with the ability to target tumor
cells and virus-infected cells(3, 4) . The initial
events involved in TNF-induced cell death occur through the 55-kDa TNF
receptor (5, 6) with the subsequent signal
transduction events being the subject of intense
investigation(7, 8) . However, the specific cellular
death pathway(s) triggered by TNF and their relationships to other
effector-induced apoptotic cell death mechanisms are not well
understood.
An evolutionary approach to the elucidation of
mechanisms involved in cell death has led to the finding that cell
death can be mediated, at least in part, by proteinases. The cell death
gene, ced-3 from Caenorhabditis elegans, and its
human homologue, the cysteine proteinase interleukin 1 converting
enzyme (ICE), cause cell death when expressed in rodent
fibroblasts(9) . Serine proteinases are implicated in cell
death induced by TNF, in that low molecular weight serine proteinase
inhibitors protect tumor cells from the cytotoxic effects of
TNF(10, 11) . More recently, the serine proteinase
inhibitor (serpin), plasminogen activator inhibitor type-2 (PAI-2), was
reported to confer protection from TNF-mediated cytolysis when
overexpressed in HT1080 fibrosarcoma cells(12) . PAI-2 is a
major product of monocytes and macrophages in response to inflammatory
mediators(13, 14) . PAI-2 was originally described as
an inhibitor of the serine proteinase, urokinase-type plasminogen
activator (uPA)(15, 16) . uPA specifically catalyzes
the hydrolysis of a single Arg-Val bond in the widely distributed
zymogen, plasminogen, to form plasmin, a broad spectrum serine
proteinase that is capable of hydrolyzing a number of protein
substrates(17) . uPA has a well established role in cellular
invasion and is involved in degradation of extracellular matrices and
tissue remodelling(17, 18) . uPA has also been
reported to function as a cellular growth factor (19, 20) and to participate in the activation of
hepatocyte growth factor(21) , but its potential role in cell
death is not known. In HT1080 tumor cells, uPA and PAI-2 are
synthesized constitutively(12) , and it is not possible to
ascertain from this model whether the presence of uPA is intrinsic to
the protective effect demonstrated by overexpression of PAI-2. In the
present paper, we report that expression of PAI-2 protects from
TNF-induced apoptosis in HeLa cells, a cell line that does not
synthesize PAI-2 or significant levels of uPA and that is sensitive to
the cytotoxic effects of TNF. Protection by PAI-2 is independent of
extracellular uPA activity and is likely to involve an intracellular
cell death proteinase.
The P site mutant
of PAI-2 was obtained by polymerase chain reaction amplification and
overlap extension following the method of Ho et
al.(24) . The following synthesized oligonucleotides were
used to alter the nucleotides at 1186-1192, resulting in a
mutation at Arg-380 (to Ala-380), and to generate a unique Nar1 restriction enzyme site (see Fig. 6A):
Primer A, 5`-GGCTCAGATTCTAGAACTCC-3`; Primer B,
5`-GTATTTCTAGAAATGCACATAAC-3`; Primer C, 5`-GACAGGCGCCACTGGACATGG-3`;
and Primer D, 5`-CCATGTCCAGTGGCGCCTGT-3`. Primers A and B and Primers C
and D, respectively, were combined to generate DNA fragments containing
overlapping mutations at nucleotides 1186-1192 in the PAI-2 cDNA
gene. These DNA fragments were then combined, and Primers A and D were
used to generate a 988-bp DNA fragment extending between the XbaI restriction enzyme sites at positions 790 and 1778 of
PAI-2 cDNA. This DNA fragment was cloned into the corresponding XbaI sites of pJ7/PAI-2 following excision of the 988-bp DNA
fragment containing the native sequence at the P
site. The
sense orientation was identified by restriction enzyme mapping, and the
DNA sequence of the mutant construct was verified by DNA sequence
analysis.
Figure 6: Protection by PAI-2 is independent of uPA. Immunoblot analysis of protein lysates from the PAI-2-expressing clone, S1a, probed with an anti-PAI-2 monoclonal antibody (A) or an anti-uPA monoclonal antibody (B) either untreated (lane 1), following treatment for 4 h with 10 ng/ml TNF (lane 2), 10 µg/ml cycloheximide alone (lane 3), or 10 ng/ml TNF and 10 µg/ml cycloheximide (lane 4). Positive controls are MM6 (5 µl of lysate from resting MonoMac6 cells) and uPA (2 ng of purified uPA standard protein). Both blots are overexposed to allow detection of possible minor bands (ECL exposure times: A, 2 min; B, 10 min). C, effect of uPA inhibitors on survival. PAI-2-expressing sense, S1a, and antisense, A2/7, transfectants were either untreated (black bars) or pretreated with the following inhibitors 30 min prior to TNF challenge: 0.1 mM amiloride (left); 0.1 mg/ml glutamyl-glycyl-arginyl-chloromethyl ketone peptide inhibitor (GGA-CK, left middle); anti-uPA monoclonal antibody (anti-uPA, right middle) at 2.5 µg/ml (shaded bars) or 5.0 µg/ml (white bars); or 1.5 µM 2,7-bis(4-amidinobenzylidene)-cycloheptan-1-one dihydrochloride (ACD; tPA Stop, American Diagnostica, right). Survival of cells was measured after 8 h by crystal violet and MTT assays and is represented as the percentage of survival relative to identical samples treated with cycloheximide alone. Treatment of the cells with these agents alone or in combination with TNF or cycloheximide had no significant effect on cell survival over the period of the assay.
For the experiments involving addition of anti-TNF or anti-uPA antibodies, the cells were washed three times in serum-free RPMI 1640, and the antibodies were added in RPMI 1640 containing 10% acid-treated fetal calf serum at the concentrations given in the figure legends. Cells were incubated for 30 min in the presence of antibody prior to treatment with TNF and/or cycloheximide. Competitive (#042) and noncompetitive (#047) anti-TNF monoclonal antibodies were obtained from Peptide Technology Ltd. (Sydney, Australia).
Figure 1:
Characterization
of transgene expression in selected stably transfected clones. A, Northern blot analysis of mRNA levels. Total RNA was
isolated from approximately 1 10
cells derived from
each of the cloned transfectants and analyzed by Northern blot using a
P-labeled probe derived from PAI-2 cDNA. The lower
panel demonstrates the total amount of RNA loaded in each lane
following reprobing of the blots for 18S rRNA. The prefix S designates sense PAI-2 transfectants, and A designates
antisense PAI-2 transfectants. CAT designates transfectants stably
transfected with the CAT cDNA gene. B, immunoblot and ELISA
analyses of PAI-2 protein expression. PAI-2 protein was detected by
immunoblot analysis of cell lysates probed with anti-PAI-2 monoclonal
antibody as described in the text. CON designates 5 µl of
lysate from lipopolysaccharide-treated MonoMac6 cells. The level of
PAI-2 in each lysate sample was quantitated by ELISA using the PAI-2
Immunobind kit and are given below each lane. The
concentration of PAI-2 is expressed as ng of PAI-2 (relative to the
PAI-2 standard provided in the kit)/mg of cellular protein (determined
by Bio-Rad protein assay). C, immunoblot analysis of CAT
protein. CAT protein of 80 kDa was detected in cell lysates from CAT
transfectants using an anti-CAT monoclonal antibody. D, uPA
binding activity of PAI-2 synthesized by transfectants. Cell lysate
from sense clone S1a was prepared as described under ``Materials
and Methods'' except that 4-(2-aminoethyl)benzenesulfonylfluoride
was omitted from the lysis buffer. To the lysate was added purified
high molecular mass uPA (55 kDa) (Calbiochem), and the mixture was
incubated for 1 h at 4 °C prior to separation of lysate proteins by
SDS-gel electrophoresis and immunoblotting. The 47-kDa PAI-2 and the
characteristic SDS-resistant uPA-PAI-2 complex (approximately 92 kDa)
were monitored using an anti-PAI-2 monoclonal antibody. Lane
1, control in the absence of added uPA; lane 2, S1a
lysate with 2 ng of uPA; lane 3, S1a lysate with 4 ng of
uPA.
The PAI-2 synthesized by the cells was tested for biological activity demonstrated by the ability to react with uPA, the serine proteinase target of PAI-2. Interaction of uPA and PAI-2 results in the formation of an SDS-resistant complex that demonstrates altered mobility on SDS-polyacrylamide gel electrophoresis or a characteristic ``band shift'' from 47 kDa to approximately 92 kDa(34) . As demonstrated in Fig. 1D, addition of purified uPA to cell lysates derived from PAI-2 transfectants results in such a band shift, demonstrating that the recombinant PAI-2 is biologically active.
Figure 2:
Sensitivity of HeLa transfectants to
TNF-mediated cytolysis. A, cell survival following treatment
with TNF and cycloheximide measured by MTT assay. The HeLa
transfectants, sense PAI-2 (S1a, S1b), antisense PAI-2 (A2/7, A2/17),
vector alone (CMV), CAT, and parental HeLa cells were assayed untreated (dotted bar) or following treatment with 10 ng/ml TNF (white bar), 10 µg/ml cycloheximide (hatched
bar), or both of these agents (black bar) for 8 h. The
data are representative of at least five independent experiments and
are expressed as the percentages of survival relative to untreated
cells. The error bars represent the standard error. Absence of
error bars indicates that the error was too small to allow display of
the error in those columns. B, cell survival following
treatment with TNF and interferon- measured by MTT assay. The HeLa
transfectants, sense PAI-2 (S1a, S1b), antisense PAI-2 (A2/7, A2/17),
vector alone (CMV), CAT, and parental HeLa cells were assayed untreated (dotted bar) or following treatment with 10 ng/ml TNF (white bar), 10 µg/ml interferon-
(hatched
bar), or both of these agents (black bar) for 8 h. As an
increase in cell numbers was observed in untreated cells over the
course of 72 h, the percentage of relative survival shown is a measure
of cell growth of treated cells after 72 h relative to the growth of
untreated cells. The data are representative of at least five
independent experiments, and the error bars represent the
standard error. C, relative survival of HeLa transfectants
with increasing TNF concentration. Each transfectant was treated with
increasing concentrations of TNF in the presence and the absence of 10
µg/ml cycloheximide and with cycloheximide alone for 8 h and
assayed for cell survival by crystal violet staining. The data are
expressed as the percentages of survival relative to cells treated with
cycloheximide alone. PAI-2-expressing sense transfectants, S1a
(
), S1b (
), S1n (
), S1/4 (
), S1/5 (⊞),
and S1/6 (
), antisense transfectant A2/7 (
), the parental
HeLa cell (
). and the CMV vector control transfectant (
). D, plot of the log of the concentration of PAI-2 expressed by
the PAI-2 sense transfectants versus the concentration of TNF
required for 50% cell death. Values are derived from the data given in (C). The symbols for each sense clone are as in C,
and the level of PAI-2 expressed by each transfectant was determined by
ELISA relative to total cellular protein as described in Fig. 1.
The line represents the best fit to the data points with a
correlation coefficient r
=
0.947.
The effect of TNF concentration on the sensitivity of the HeLa transfectants to cell death is shown in Fig. 2C. The PAI-2-expressing cell lines show enhanced survival at concentrations of TNF below 10 ng/ml but demonstrate less resistance to the cytolytic effects of TNF at higher concentrations of TNF. At 10 ng/ml the clones expressing the highest levels of PAI-2, S1a and S1b, were essentially resistant to TNF-induced cell death, as compared with antisense PAI-2, CAT, or CMV transfectants; whereas at 50 ng/ml TNF, S1a and S1b show approximately 50% survival compared with 25-30% for the control transfectants. Furthermore the resistance to TNF-induced cell death appears to correlate with the relative level of PAI-2 expressed by the transfected cells. Indeed, when the concentration of TNF required for 50% cell death for each of the sense PAI-2-expressing cell lines was plotted against the log of the concentration of PAI-2 expressed by each cell line (Fig. 2D), a linear relationship was obtained.
That the cell death observed was a direct consequence of TNF action was demonstrated by comparing the effects of noncompetitive and competitive anti-TNF antibodies (which interfere with TNF binding to its receptor) on cell death (Fig. 3A). The PAI-2-expressing clone, S1a, and the antisense clone, A2/7, were incubated with increasing concentrations of each of the antibodies and then subjected to challenge with TNF and cycloheximide. Incubation with increasing concentrations of the competitive anti-TNF antibody resulted in increased survival of both the sense and the antisense clones, whereas the noncompetitive antibody had no significant effect on the relative survival of either clone. Therefore, PAI-2 demonstrates protection against cell death mediated directly through TNF.
Figure 3:
PAI-2
protects against cell death mediated by TNF, but protection is not
correlated with TNF receptor expression. A, effect of
competitive and noncompetitive anti-TNF antibodies on cytolysis by TNF
of PAI-2 transfectants. The PAI-2 sense transfectant S1a () and
the antisense transfectant A2/7 (
) were preincubated with
increasing concentrations of anti-TNF antibodies either competitive for
TNF binding (solid line) or noncompetitive for TNF binding (dotted line) for 30 min. Cells were then treated with 10
ng/ml TNF and 10 µg/ml cycloheximide as described under
``Materials and Methods.'' Cell survival was measured by MTT
assay and is represented as the percentage of survival relative to an
identical sample treated with cycloheximide alone. Treatment of the
cells with antibodies alone or in combination with TNF or cycloheximide
had no significant effect on cell survival over the period of the
assay. B, expression of 55-kDa TNF receptor in HeLa cell
transfectants with differential sensitivity to TNF cytolysis measured
by Northern blot analysis. Total RNA was isolated from approximately 1
10
cells derived from each of the cloned
transfectants and analyzed by Northern blot using a
P-labeled probe derived from 55-kDa TNF receptor cDNA.
HeLa transfectants are as indicated, and CON designates mRNA
isolated from MonoMac6 control cells. The lower panel demonstrates the total amount of RNA loaded in each lane following
reprobing of the blots for 18 S rRNA. The bar graphs represent
the levels of 55-kDa TNF receptor mRNA quantitated from scanned
autoradiographs, normalized to the 18 S rRNA loading control and
expressed relative to the MonoMac6 mRNA control (CON) loaded
on each gel. C, TNF-induced expression of manganese superoxide
dismutase mRNA in HeLa cell transfectants with differential sensitivity
to TNF cytolysis. Total RNA was isolated from approximately 1
10
cells derived from each of the HeLa transfectants and
analyzed by Northern blot using a
P-labeled probe derived
from manganese superoxide dismutase cDNA. The lower panel demonstrates the total amount of RNA loaded in each lane following
reprobing of the blots for 18 S rRNA. D, electrophoretic
mobility shift analysis of NF
B activation in nuclear extracts from
HeLa clones following treatment with TNF. The arrow indicates
the retarded complex containing NF
B, which is abolished in the
presence of cold NF
B competitor DNA. NS denotes
nonspecific binding.
Figure 4:
Expression of manganese superoxide
dismutase and bcl-2 in HeLa cell transfectants with
differential sensitivity to TNF cytolysis. A, Northern blot
analysis of manganese superoxide dismutase mRNA levels in PAI-2
transfectants. Total RNA was isolated from approximately 1
10
cells derived from each of the cloned transfectants and
analyzed by Northern blot using a
P-labeled probe derived
from manganese superoxide dismutase cDNA. The Northern blots are
overexposed to reveal the constitutive low levels of manganese
superoxide dismutase mRNA. The lower panel demonstrates the
total amount of RNA loaded in each lane following reprobing of the
blots for 18 S rRNA. The bar graphs represent the levels of 4-
and 1-kb manganese superoxide dismutase mRNA quantitated from scanned
autoradiographs, normalized to the 18S rRNA loading control and
expressed relative to the MonoMac6 mRNA control (CON) loaded
on each gel. B, immunoblot analysis of bcl-2 protein
expression in PAI-2 transfectants. Cell lysates from each of the PAI-2
transfectants was probed with anti-bcl-2 monoclonal antibody as
described in the text. For both A and B, the PAI-2
sense transfectants are S1a, S1b, S1/4, S1/5, and S1/6, and the
antisense PAI-2 transfectants are A2/7, A2/12, A2/15, A2/16, and A2/17.
HeLa is the parental cell; CON is lipopolysaccharide treated MonoMac6
cells. ASM1 is a transfectant containing Ala-380 PAI-2 (see Fig. 1).
Figure 5: Expression of PAI-2 protects from apoptotic cell death. Fluorescent micrographs of Hoechst (No. 33258) stained nuclei from the antisense A2/7 (A and B) and the PAI-2-expressing sense S1a (C and D) transfectants either untreated (A and C) or following treatment with 10 ng/ml TNF and 10 µg/ml cycloheximide for 4 h (B and D). E, electrophoretic separation on agarose of DNA isolated from nuclei of PAI-2 sense (S1a), antisense (A2/7), HeLa parental cells, and a transfectant containing Ala-380 PAI-2 (ASM1) either untreated(-) or following treatment with TNF and cycloheximide for 6 h (+). The gel was visualized by staining with ethidium bromide.
The ability of PAI-2 to block
TNF-induced apoptosis appears to be specific, in as much as the PAI-2
transfectants are susceptible to other stimuli that induce apoptotic
cell death in HeLa cells, such as ultraviolet (UVC, 30
J/m) or gamma (1 kiloradian) irradiation (data not
shown).
Figure 7:
Mutation of the PAI-2 serpin P residue abolishes the protective effect of PAI-2. A,
diagram indicating the mutations engineered into the PAI-2 cDNA
sequence to convert the P
-arginine to an alanine in the ASM
construct (Ala(380) PAI-2). B, relative levels of
mutant PAI-2 in ASM1 and ASM2, compared with wild-type PAI-2 in S1a,
S1n, and S1/5. HeLa cells were transfected with the ASM construct,
stable transfectants were isolated, and PAI-2 expression was monitored
in cell lysates by immunoblot analysis. The mutant PAI-2 does not
demonstrate uPA binding activity as demonstrated by preincubation of
ASM1 cell lysate with 2 ng of 55-kDa uPA for 1 h as described in the
legend to Fig. 1D. Blots were probed with an anti-PAI-2
monoclonal antibody. C, relative survival of stable
transfectants expressing wild-type and mutant PAI-2. Each of the
transfectants was treated with an increasing concentration of TNF, in
both the presence and the absence of 10 µg/ml cycloheximide,
treated with cycloheximide alone for 8 h, and assayed for cell survival
by crystal violet staining. The data are represented as the percentage
of survival relative to identical samples treated with cycloheximide
alone. Treatment with either cycloheximide alone or TNF alone at the
concentrations shown had no significant effect on cell survival over
the period of the assay. S1a (
), S1/5 (
), ASM1 (┘),
ASM2 (|oT), S1n (
), and A2/7 (
)
transfectants.
TNF is a potent inducer of cell death in many systems, and
some cells have developed mechanisms to protect against the cytotoxic
effects of TNF. The results of this study show that expression of the
macrophage product, PAI-2, protects HeLa tumor cells from apoptosis
induced by TNF. The apoptotic death pathway blocked by PAI-2 is
independent of bcl-2 and manganese superoxide dismutase. The
mechanism of PAI-2 protection is independent of uPA but requires the
P-Arg amino acid residue of PAI-2.
The findings that
serine proteinases may be involved in TNF-induced cell death (10, 11) and that PAI-2 biosynthesis is part of an
acute phase cellular response to TNF (49) has led to an
investigation of the possible function of PAI-2 in TNF-mediated
cytotoxicity. Kumar and Baglioni (12) provided the first
evidence that PAI-2 may function as a protective protein in TNF-induced
cytotoxicity by demonstrating that overexpression of PAI-2 in the
fibrosarcoma cell line HT1080 conferred resistance to these cells from
the cytotoxic effects of TNF. We demonstrate that expression of PAI-2
in cells that do not synthesize endogenous PAI-2 confers protection
from TNF-induced cell death and that PAI-2 protects against apoptosis.
Protection from TNF-mediated apoptosis is observed in the presence of
either the protein synthesis inhibitor cycloheximide or the
inflammatory cytokine, interferon-. Several PAI-2-expressing
clones were assayed for their level of PAI-2 expression, and this was
correlated with their protection against TNF. Measurement of PAI-2
levels by ELISA shows that the levels of PAI-2 required for protection
are well within the range produced by nontransfected cells.
lipopolysaccharide-induced macrophage-like MonoMac6 cells synthesize a
4-fold greater amount of PAI-2 than the transfected cell line
expressing the highest levels of PAI-2. A threshold level of PAI-2
appears to be required to protect cells against TNF-induced cell death
with increases beyond this threshold having no additional protective
effect.
TNF-binding proteins, which are soluble extracellular domains of TNF receptors produced by proteolytic cleavage, can be actively shed by cells and may function to prevent TNF from inducing continuous damage to the cell(38) . The reduced sensitivity of PAI-2 transfectants to TNF is unlikely to be due to such altered TNF-receptor binding in these cells, because the sensitivity of the different transfectants to TNF could not be correlated to loss of TNF receptors or impaired ability of TNF to bind to receptors. Furthermore, signal transduction through the 55-kDa TNF receptor functions similarly in transfected cells as in control cells, evidenced by the TNF-mediated transcriptional activation of manganese superoxide dismutase, indicating that this TNF signaling pathway is functional in these cells.
Inhibition of protein synthesis in many tumor cells increases their sensitivity to TNF, an observation that has led to the suggestion that TNF itself may induce ``protective proteins'' in TNF-resistant cells. Overexpression of the oxygen radical scavenger manganese superoxide dismutase partially protects from TNF cytotoxicity, suggesting that other TNF-inducible proteins exist that are involved in conferring protection(40) . The oncogene bcl-2 protects from apoptotic cell death but appears to have a cell-specific effect against TNF-induced cell death(41, 50) . The levels of manganese superoxide dismutase and bcl-2 were not found to correlate with expression of PAI-2 nor with the sensitivity of each of the transfectants to TNF-induced apoptosis. Given that manganese superoxide dismutase protects from cell death mechanisms mediated by oxygen radicals (51) and that PAI-2 does not protect from apoptosis induced by ultraviolet or ionizing radiation, both of which utilize oxygen radicals in their cytolytic mechanisms(52) , it is likely that PAI-2 exerts its effect either upstream or independently of manganese superoxide dismutase. Thus, there may be several pathways leading to cell death, which possibly converge at different points, and it seems likely that several proteins may inhibit different arms of the cell death response.
The mechanism by which PAI-2 confers resistance
to TNF cell death is not yet clear but is unlikely to be associated
with uPA proteolytic activity. The data presented do not support a role
for uPA in TNF-mediated cell death and further suggests that an
intracellular proteinase target for PAI-2 might exist. Members of the
serpin superfamily, such as PAI-2, commonly act as proteinase
inhibitors, and there are precedents of serpins interacting with
multiple proteinase targets. There is also indirect evidence for the
involvement of proteinases in TNF-mediated cell
death(10, 11) . A characteristic of serine proteinases
is the catalytic triad of Ser, His, and Asp residues that form a
hydrogen bonding system responsible for peptide bond cleavage. The
substrate specificity of serine proteinases is determined by
interaction at the primary substrate binding site (S).
Serine proteases can be divided into three major groups based on their
S
specificity(53) : trypsin-like enzymes, which
cleave substrates with positively charged residues such as Arg or Lys
at the P
site due to interaction with Asp in the S
pocket of these enzymes; chymotrypsin-like enzymes, which cleave
substrates with aromatic or large hydrophobic residues at the
P
position; and elastase-like proteinases, which prefer
substrates with small aliphatic residues at the P
position.
The essential requirement for an Arg in the serpin P
site
of PAI-2 is evidence for the involvement of a proteinase and possibly a
trypsin-like serine proteinase in the apoptotic death pathway blocked
by PAI-2. A further implication of this finding is that the proteinase
is likely to have a sequence preference for Arg adjacent and
amino-terminal to the scissile peptide bond (P
residue) of
its natural substrate. Identification of an intracellular proteinase
that is inhibited by PAI-2 may provide a key to unravelling a novel
pathway leading to cell death.
Alternatively, PAI-2 may inhibit an
Arg-specific cysteine proteinase involved in TNF-induced apoptosis. A
proteinase recently associated with the regulation of cell death and
apoptosis is the cysteine proteinase, ICE. A specific inhibitor of ICE
is the cowpox virus encoded serpin, crmA(54) . PAI-2
and crmA show a high degree of structural similarity; residues
46-338 of crmA share 35% sequence identity with residues
110-415 of PAI-2. ()However, PAI-2 is unlikely to
represent the mammalian homologue of crmA, because crmA contains an Asp in the P
residue, and there is a
requirement for a P
-Asp in the catalytic mechanism of
ICE(55, 56) . ICE-like activity was found to be
similar in PAI-2-expressing and -nonexpressing cells (see
``Material and Methods''), indicating that PAI-2 does not
inhibit ICE-like proteinases. Other serpins have been described that
contain Arg in the P
position, which include maspin, a
tumor suppressor gene implicated in breast cancer(57) ; the
vaccinia virus-encoded protein K2L(58) ; the Limulus encoded intracellular coagulation inhibitor(59) ; and the
myxoma virus-encoded SERP-1(60) . Further studies are required
to determine the relationships between the functions and specific
biological targets of these serpins and those of PAI-2.
The results presented here illustrate a function for intracellular PAI-2 in cell death, which may have important implications in the resolution of inflammatory and cell-mediated immune reactions that are mediated by TNF. Monocytes rely on uPA activity on their cell surface to affect invasion of tissues to access various targets, for example, inflammatory sites. PAI-2 has been shown to function extracellularly to limit uPA activity and thus regulate the invasive capabilities of monocytes(61) . The vast majority of PAI-2 synthesized by monocytes, however, remains intracellular, probably due to an inefficient signal sequence(62) . Activation of monocytes by inflammatory agents such as TNF induces a rapid accumulation of high levels of PAI-2 in the cytoplasm that does not appear to be correlated with uPA synthesis (63) and may play a functional role in regulating apoptotic cell death in these cells. It is of note that a PAI-2 proteolytic cleavage product has recently been linked with apoptosis in myeloid leukemic cells(64) , suggesting that loss of the apoptosis inhibitory activity of PAI-2 may be associated with proteolytic cleavage of the PAI-2 molecule.
Monocytes at sites of inflammation may be exposed to concentrated doses of cytokines, including TNF, which may in combination be cytotoxic. Intracellular PAI-2 may be a part of a cellular response of monocytes/macrophages to delay cell death in order to ensure that they survive secretion of their own TNF, possibly to allow them to complete vital functions such as lymphocyte activation and antigen presentation prior to apoptosis. In support of the role of PAI-2 in cell death, PAI-2 is expressed in vivo in skin (65) in a zone associated with keratinocyte differentiation and apoptosis. Tumor cells have also been found to synthesize PAI-2(66, 67) , and transformation by ras and src oncogenes has been shown to induce PAI-2 expression in vitro(68) . Given the effect of PAI-2 on cell survival, acquisition of PAI-2 expression by tumor cells may reduce their susceptibility to TNF-mediated apoptosis and thereby allow a selective survival advantage to these tumor cells.