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Department of Surgery, Division of Hospital Medicine, University of Bristol, Bristol Royal Infirmary, Bristol, United Kingdom BS2 8HW
Address all correspondence and requests for reprints to: Dr. Laura A. Maile, Division of Endocrinology and Metabolism, Department of Medicine, CB no. 7170, 6111 Thurston Bowles Building, University of North Carolina, Chapel Hill, North Carolina 27510-7170
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Abstract |
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In this study we confirmed that preincubation of Hs578T cells with IGFBP-3 enhances the apoptotic effect of the ceramide analog C2. The presence of IGF-I completely inhibited the enhancement effect, apparently by inhibiting cell surface association and proteolytic modification. The presence of a serine protease inhibitor [4-(2-aminoethyl)benesulfonyl fluoride] completely inhibited the enhancement effect of IGFBP-3, and Western immunoblotting of conditioned medium and cell surface-associated IGFBP-3 revealed that proteolytic fragmentation of the IGFBP-3 was reduced. In addition, fragments from the incubation of IGFBP-3 with plasmin were able to enhance the susceptibility of Hs578T cells to C2. The effect of these fragments could, however, also be reduced by 4-(2-aminoethyl)benesulfonyl fluoride despite the fact that IGFBP-3 was already fragmented. This suggests additional roles for serine proteases in the IGFBP-3 effect on C2-induced apoptosis in addition to the cleavage of the binding protein.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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There is increasing evidence, however, to support additional, IGF-independent roles for IGFBP-3. We have reported recently that IGFBP-3 can significantly increase the apoptotic effect of C2, a ceramide analog and physiological trigger of apoptosis, on Hs578T breast carcinoma cells (2). As these cells lack functional IGF receptors, do not secrete measurable amounts IGF-I or IGF-II, and are nonresponsive to added IGF, they provide an excellent model for studying the IGF-independent effects of IGFBP-3.
Specific binding sites for IGFBP-3 have been demonstrated on Hs578T cells, and IGFBP-3 has been shown to inhibit growth and DNA synthesis in these cells, presumably by interacting with these cell surface binding sites (3, 4). This inhibitory effect is, however, reversed by coincubation with IGF-I or IGF-II, and this reversal was attributed to the formation of IGFBP-3/IGF complexes in which IGFBP-3 cannot bind to the cell surface and is protected from proteolysis (5).
Proteolysis of IGFBP-3 with a subsequent reduction in affinity for the IGFs has been described as a mechanism for increasing IGF availability to the tissues and thereby negating the inhibitory effect of IGFBP-3 on IGF action. We have previously shown that in contrast to the circulation, in extravascular fluids, such as interstitial (6) and synovial (7) fluids, almost all of the IGFBP-3 is in a fragmented form. More recently, a fragment of IGFBP-3 that apparently does not bind to IGF has been shown to have specific inhibitory effects on cell growth in PC-3 cells (8).
The aim of the experiments described here was to study the role of proteolysis in modulating the effect of IGFBP-3 on C2-induced apoptosis. The enhancement effect of IGFBP-3 on C2-induced apoptosis was completely inhibited by the addition of IGF-I, and this appeared to be due to the formation of IGF-IGFBP-3 complexes that could not associate with the cell surface and that protected IGFBP-3 from proteolysis. The enhancement effect of IGFBP-3 on C2-induced apoptosis was also completely inhibited in the presence of a noncytotoxic serine protease inhibitor, 4-(2-aminoethyl)benesulfonyl fluoride (AEBSF). The presence of this inhibitor, however, only partially affected the cleavage of the binding protein. In addition, prefragmented IGFBP-3 enhanced C2-induced apoptosis, and this effect was partially blocked by the presence of the serine protease inhibitor. This study demonstrates that both intact and already fragmented IGFBP-3 can increase the susceptibility of Hs578T cells to apoptosis. It also suggests the involvement of at least one serine protease in the enhancement effect of IGFBP-3, but suggests the involvement of serine protease activity that is modulated by IGFBP-3 but does not necessarily result in direct cleavage of IGFBP-3.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture
Hs578T cells, an estrogen receptor-negative breast carcinoma
cell line, was purchased from European Collection of Animal Cell
Cultures (Porton Down, UK) and maintained in 75-cm2
flasks in a humidified atmosphere of 5% CO2 at 37 C. Cells
were grown in DMEM with Glutamax-1 (Gibco Life Technologies, Paisley, UK) supplemented with 10% FCS,
penicillin (5000 IU/ml), and streptomycin (5 mg/ml; growth medium). For
each experiment cells were seeded in six-well plates at a density of
0.1 x 106 cells/well and cultured in growth medium
for 24 h. Cells were then cultured for 24 h in phenol
red-free, serum-free HEPES-DMEM and Ham’s nutrient F-12 (SFM) with
sodium bicarbonate (0.12%), BSA (0.2 mg/ml), transferrin (0.01 mg/ml),
penicillin (5000 IU/ml), and streptomycin (5 mg/ml).
Effects of AEBSF and IGF-I on IGFBP-3 enhancement of C2-induced
apoptosis
Cells were incubated with 100 ng/ml recombinant ngIGFBP-3 for
24 h either alone or with AEBSF (0.1 mM) or IGF-I (50
ng/ml). This was followed by a further 24-h incubation with 100 ng/ml
IGFBP-3 and 7 µM C2 alone, together, and in combination
with AEBSF (0.1 mM) or IGF-I (50 ng/ml).
Effect of fragments of IGFBP-3 generated by the incubation of
ngIGFBP-3 with plasmin on C2-induced apoptosis
The ngIGFBP-3 was incubated with plasmin (1:1, wt/wt) for 3
h at 37 C in SFM. The incubation was then heated to 56 C to inactivate
the protease. Cells were then incubated with 100 ng/ml
plasmin-fragmented IGFBP-3 (pIGFBP-3) for 24 h either alone or
with AEBSF (0.1 mM), followed by a further 24-h incubation
with 100 ng/ml pIGFBP-3 and 7 µM C2 alone, together, and
in combination with AEBSF.
Cell death assessment and confirmation of apoptosis
Floating cells and attached cells dispersed with trypsin-EDTA
were pelleted from the conditioned medium (CM) from each treatment and
the CM was stored at -20 C. Cell pellets were washed with PBS and then
resuspended in 1 ml PBS. Cell death was assessed by staining a 50-µl
aliquot with trypan blue (1:1), and dead cells were measured by their
failure to exclude the dye and expressed as a percentage of the total
cells.
After removal of an aliquot for cell counting, the cells were repelleted and fixed in 1 ml 70% ethanol for at least 24 h at 4 C. The fixed cells were then pelleted again and washed three times in PBS. Supernatant was removed, and cells were resuspended in reaction buffer (propidium iodide, 0.05 mg/ml; sodium citrate, 0.1%; ribonuclease A, 0.02 mg/ml; Nonidet P-40, 0.03%; pH 8.3) and incubated at 4 C for 30 min before measurement on a FACSCalibur flow cytometer (Becton Dickinson and Co., Mountain View, CA) with an argon laser at 488 nm for excitation. Apoptotic cells with a lower DNA content have less staining than normal cells and appear as a pre-G1 peak on a DNA cell cycle histogram. The data were analyzed using a CellQuest software package (Becton Dickinson).
Western immunoblotting of CM
Samples of CM were boiled for 5 min in SDS-containing
sample buffer before loading on a 12.5% SDS-polyacrylamide gel, which
was run overnight at 35 mA. Proteins were then transferred by
electrophoresis at 70 mA onto Hybond C membranes for 4 h at 0.8-mA
constant current. Intact and fragmented ngIGFBP-3 was visualized by
Western immunoblotting. The membranes were blocked in Tris-buffered
saline and 3% nonfat milk before incubation with either rabbit
antiintact IGFBP-3 (SCH 2/6 at 1:10,000 dilution) or sheep
antisynthetic peptide at room temperature overnight. The membranes were
then washed in Tris-buffered saline to remove unbound antibody before
incubating with either an antirabbit (1:10,000) or antisheep antibody
(1:2,000; DAKO Corp., Carpenteria, CA) conjugated to
peroxidase for 1 h at room temperature. Binding of the
peroxidase-labeled antibody was visualized using enhanced
chemiluminesence with an ECL detection system (Amersham Pharmacia Biotech, Aylesbury, UK) and exposure to x-ray film.
Band intensities on autoradiographs were measured by scanning densitometry (Bio-Rad Laboratories, Inc., Hercules, CA) and analyzed using Molecular Analyst software (Bio-Rad Laboratories, Inc.).
Visualization of cell surface-associated and internalized
IGFBP-3
Cells were incubated as previously described. The CM was
removed, and cells were washed briefly with PBS before adding 2 ml 0.1
M acetic acid to each well. The six-well plates were
incubated for 30 min at 4 C. The acetic acid was collected, frozen, and
replaced with 125 µl lysis buffer [10 mM Tris-HCl (pH
7.6), 5 mM EDTA, 50 mM NaCl, 30 mM
sodium pyrophosphate, 50 mM sodium fluoride, and 100 µm
sodium orthovanadate] for 15 min on ice. The cells were scraped from
the wells and frozen. IGFBP-3 in the cell washes and lysates was
visualized by Western immunoblotting as previously described.
In vitro protease assay of CM
Samples of CM were assayed for their ability to fragment
radiolabeled ngIGFBP-3 ([125I]ngIGFBP-3 iodinated using a
chloramine-T method) according to the method of Lamson et
al. (9). Briefly, 50 µl of each CM sample were incubated with
15,000 cpm [125I]ngIGFBP-3 for 24 h at 37 C. The
assay was stopped by boiling 20-µl aliquots of the incubation with
SDS loading buffer before loading onto a 12.5% SDS-polyacrylamide gel,
which was run overnight at 30 mA. Fragmentation of the labeled
substrate was visualized by fixing and drying the gels and exposing
them to x-ray film at -70 C for 2–3 days.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Inhibition of the enhancement effect of IGFBP-3 on C2-induced
apoptosis
Figure 1A
shows the percentage of
dead cells in each treatment, as assessed by trypan blue counts, and
Fig. 1B shows the percentage of cells appearing as a pre-G1
peak (apoptotic), as measured by flow cytometry. Essentially, the same
pattern of results was obtained with both techniques. The level of
apoptosis in SFM was not affected by incubation with ngIGFBP-3. The
percentage of cells undergoing apoptosis was significantly increased,
however, when cells were cultured in the presence of C2. Preincubation
with ngIGFBP-3 before challenge with C2 significantly increased
apoptosis, confirming our previous report. The presence of AEBSF, the
noncytotoxic serine protease inhibitor, had no significant
effect on apoptosis levels after SFM, C2, or ngIGFBP-3
treatment alone. However, the presence of AEBSF completely
inhibited the enhancing effect of ngIGFBP-3 on C2-induced
apoptosis; levels of apoptosis were comparable to those with C2
alone.
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, A and B, also shows cell death,
as measured by trypan blue, and apoptosis, as measured by flow
cytometry. Figure 2 shows that the presence of IGF-I completely
inhibited the enhancement effect of ngIGFBP-3. Levels of apoptosis in
the presence of IGF-I, ngIGFBP-3, and C2 were again reduced to that
seen after C2 alone. We have previously shown that IGF-I alone has no
direct survival effect in these cells (2).
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, A and B, shows that incubation
with pIGFBP-3 had no significant effect on the level of apoptosis
alone, but, similar to ngIGFBP-3, significantly enhanced the apoptotic
effect of C2. This figure also shows that although the levels of
apoptosis in the presence of C2 plus pIGFBP-3 and AEBSF were not
reduced to those in the presence of C2 alone, the enhancing effect of
pIGFBP-3 was significantly reduced in the presence of AEBSF.
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, lane 2). To ensure that proteolysis
did not merely reflect instability of ngIGFBP-3 at 37 C, ngIGFBP-3 was
incubated in SFM in the absence of cells for 24 h, and no
significant amount of proteolysis was observed (Fig. 4, lane 1). The
presence of C2 had no effect on the extent or pattern of proteolysis
(Fig. 4, lane 3). Western immunoblots from three separate experiments
were analyzed using densitometry, and results are summarized in Table 1. The presence of AEBSF significantly
reduced the amount of fragments detectable in the CM and increased the
amount of intact ngIGFBP-3 detectable in the CM (Fig. 4, lane 5,
compared with control lane 4). The presence of IGF-I also resulted in a
significant reduction in fragmentation compared with IGFBP-3 incubated
alone and a greater increase in intact ngIGFBP-3 than was seen with
AEBSF (Fig. 4, lanes 7 and 8, compared with control lane 6).
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(lane 1) shows that
preincubation of ngIGFBP-3 with plasmin resulted in the generation of
fragments of 21–25 and 16 kDa, consistent with what has been reported
previously (10). The addition of pIGFBP-3 to the cells in the presence
or absence of C2 did not result in any additional proteolysis of
pIGFBP-3 (lanes 2 and 3). The coincubation of pIGFBP-3 with AEBSF
resulted in a very faint band of intact IGFBP-3 in the CM, suggesting
that there is a small amount of intact ngIGFBP-3 remaining after the
incubation with plasmin, which is subsequently cleaved (lanes 4 and
5).
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, lane 1, compared with Fig. 4
, lanes 1
and 2). The presence of AEBSF did not have any significant effect on
cell surface association of intact ngIGFBP-3, but no fragments were
detectable (Fig. 6A, lane 3). In contrast, the presence of IGF-I
markedly reduced the amount of intact ngIGFBP-3 that was associated
with the cell surface, and in addition, there were no fragments
detectable (Fig. 6A, lane 2). Densitometric analysis of Western
immunoblots from three independent experiments showed an 88.7 ±
11.3% reduction in IGFBP-3 associated with the cell surface in the
presence of IGF-I compared with IGFBP-3 incubated alone.
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, lanes 1 and 2).
Figure 7 shows the results from a
Western immunoblot of cell lysates from cells that had been
incubated with ngIGFBP-3. Lane 1 shows that after cell surface
washing with acetic acid, only a 16-kDa fragment was detectable; lane 2
is a representative lysate from cells that had not been previously
washed with acetic acid and shows both intact and fragmented IGFBP-3
comparable to those seen on the cell surface.
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).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study we confirmed our previous report that ngIGFBP-3 added to cultures of Hs578T cells predisposes these cells to C2-induced apoptosis and that specific fragments of this added ngIGFBP-3 can be detected in the medium regardless of the presence of C2. Surprisingly, however, in an in vitro protease assay, the CM did not show any IGFBP-3 protease activity. Visualization of the cell surface-associated ngIGFBP-3 revealed that most of the ngIGFBP-3 was intact. These data led us to conclude that ngIGFBP-3 added to the medium surrounding Hs578T cells binds to the cell surface, where it is proteolytically cleaved. The larger fragments, possibly because of a reduced affinity for the cell surface, then accumulate in the CM.
Inhibition of the enhancement effect of IGFBP-3 by IGF-I would appear to be due to the formation of IGF-IGFBP-3 complexes that prevent IGFBP-3 binding to the cell surface, evident by the significant reduction in intact IGFBP-3 on the cell surface. Formation of these complexes protects IGFBP-3 from subsequent proteolysis; hence, there is an accumulation of intact IGFBP-3 in the CM.
Detection of only a 16-kDa fragment of IGFBP-3 in the cell lysates after washing the cell surface suggested that this fragment, in contrast to the intact IGFBP-3, may either be internalized or resistant to removal by washing. This fragment may, therefore, be important in the enhancement effect of IGFBP-3 on C2-induced apoptosis. Visualization of the cell surface-associated ngIGFBP-3 revealed that the presence of AEBSF had no significant effect on cell surface binding of the intact protein, but the 16-kDa fragment was not detectable. The involvement of a 16-kDa fragment is consistent with data describing the growth inhibitory effects on PC-3 cells of a 16-kDa fragment of IGFBP-3 generated from the cleavage of ngIGFBP-3 by plasmin (11). This 16-kDa fragment was N-terminal sequenced and shown to comprise amino acids 1–95 (10). Detection of this fragment with antibodies raised to amino acids 92–104 of IGFBP-3 in this study (data not shown) suggests that this 16-kDa fragment may be the same as that shown to have growth inhibitory effects on PC-3 cells.
The reduction in the level of fragments present on the cell surface may account for the inhibitory effect of both IGF and AEBSF. However, the presence of AEBSF only resulted in a relatively small reduction in proteolysis, evident by the fragments still detectable in the CM. The partial reduction of the proteolysis of IGFBP-3, yet complete abrogation of the enhancing effect on C2-induced apoptosis, suggest that although the effect of IGFBP-3 may require proteolytic modification of the binding protein to generate specific bioactive fragments, it may also involve some other serine protease action.
The plasmin-generated fragments of ngIGFBP-3 were also able to enhance the C2-induced apoptosis to a level higher than that of the ng-IGFBP-3. These data further support the idea that the enhancing effect of IGFBP-3 may be mediated via a specific fragment. However, the effect of these fragments was also reduced in the presence of AEBSF. This further supports the possibility that the effect of ngIGFBP-3 on C2-induced apoptosis not only involves proteolytic modification of the binding protein, but also required the activation of a signaling pathway in which a serine protease is required. Serine proteases have been shown recently to be essential for apoptosis induced by a number of triggers apparently upstream of ceramide generation (12, 13). Results from our laboratory, which suggest that the enhancing effect of IGFBP-3 is specific to the ceramide-activated pathway of apoptosis, would be consistent with the idea that IGFBP-3 activates a serine protease upstream of ceramide generation, which subsequently enhances the activity of this pathway (14). As it was not possible to increase levels of AEBSF so that it was completely effective, as AEBSF is toxic at higher levels, we cannot say absolutely that the effect of IGFBP-3 is due to the enhancement of an upstream protease. Our data are consistent with a serine protease being involved that is not solely due to direct cleavage of IGFBP-3. That IGFBP-3 can modulate the activity of serine proteases for which it is not itself a direct substrate has clearly been established by demonstrations that IGFBP-3 can modify the activity of an IGFBP-4-specific serine protease (15, 16). Further studies will be necessary to fully elucidate the mechanism by which IGFBP-3 enhances the susceptibility of Hs578T cells to apoptotic triggers.
In summary, we have shown that binding of IGF-I to IGFBP-3 inhibits cell surface association, subsequent proteolysis, and the enhancing effect of IGFBP-3 on C2-induced apoptosis. We have also shown IGFBP-3 has identical effects whether intact or cleaved. In addition, we have shown that AEBSF, although completely inhibiting the enhancing effect of the binding protein on C2-induced apoptosis, only partially affected its cleavage and only partially blocked the effect of pregenerated fragments. These data suggest that the enhancing effect of IGFBP-3 may not only involve proteolytic modification of the binding protein, but may also require the modulation of another serine protease directly involved in mediating the effect of IGFBP-3, although not necessarily involved in direct IGFBP-3 cleavage.
Received November 18, 1998.
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: CrmA and Bcl-2 target distinct components in the apoptotic
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