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Pediatric Endocrinology Unit, Department of Women and Child Health, Karolinska Hospital and Institute (P.B.), Stockholm Sweden; and Department of Pharmacokinetics and Metabolism, Genentech Incorporated (P.J.F.), South San Francisco, California 94080
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Introduction |
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Proteolysis of IGFBP-3 was first demonstrated in pregnancy serum from humans and rodents (4, 5, 6, 11). This proteolytic processing of IGFBP-3 results in reduced affinity of IGFBP-3 for IGFs and, in particular, for radiolabeled IGF-I (12, 13) and has been demonstrated to increase IGF bioavailability in serum (8, 9). The increased IGFBP-3 protease activity in maternal serum may only reflect the activation of proteases within a specific organ and subsequent dilution of the activity into the general circulation. Thus, the resulting release of IGF from circulating stores in the ternary complex may be more pronounced within that organ. If the major production site of the pregnancy IGFBP-3 protease activity is the placenta, as has been postulated (14), this activity may help support fetal growth by increasing maternal IGF bioavailability to the placenta. In human fetal serum, which does not display increased IGFBP-3 protease activity, an increased IGF-I bioavailability is accomplished by low fetal ALS expression resulting in lack of the ternary complex (15, 16).
The identity of the pregnancy-specific IGFBP-3 protease activity in human and rodent serum is not known, although it has been suggested to be divalent-cation dependent and inhibited by serine protease inhibitors (17, 18). In sera from pregnant rats, tissue inhibitors of matrix metallo-proteinases (TIMP-1) have recently been demonstrated to inhibit the proteolysis of IGFBP-3 (19). In the present study we partially purified the pregnancy-associated IGFBP-3 proteases from pregnant human serum (PHS). Two distinct protease activities were identified: one activity of approximate 70–90 kDa, which was present in both pregnant and nonpregnant human serum (NHS), and was further demonstrated to be plasminogen. The other activity, present only in PHS, was distinct from the 70- to 90-kDa protease because it was of larger molecular mass (>150 kDa), was unaffected by plasminogen/plasmin activators (PAs) and inhibitors, as well as serine protease inhibitors, but was inhibited by EDTA and zinc. We suggest that at least two protease activities may be involved in the regulation of IGF bioavailability in human serum, one of which is present during pregnancy.
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Materials and Methods |
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Plasminogen, plasmin, streptokinase, urokinase (UK), aprotinin, and
-2-antiplasmin, and D-PHE-PRO-ARG chloromethyl ketone (PPACK) were
purchased from Calbiochem (La Jolla, CA). Plasminogen-depleted plasma
(PDP) was purchased from American Diagnostic (Greenwich, CT). IGF-I,
IGFBP-3, and tissue PA (tPA) are products of Genentech, Inc.
IGFBP-3 protease assay
Proteolytic activity was measured as the ability of a sample to
degrade radiolabeled 125I-IGFBP-3 (20). Serum (2–3 µl),
column fractions (10–20 µl), or pure proteases were incubated with
30,000 cpm of 125I-human glycosylated IGFBP-3 (DSL,
Webster, TX) for 5 h at 37 C (± protease inhibitors) in a total
volume of 50 µl 20 mM HEPES, pH 7.2, 5 mM
Ca2+, and 0.1% BSA. Reactions were terminated by addition
of SDS sample buffer, and the reaction mixture was separated by
SDS-PAGE (12.5% or 15% gels) at 45V overnight as previously described
(15). Gels were dried and exposed to x-ray films for 1–4 days at -70
C. In addition, to check for possible substrate competition or
differences in the susceptibility for proteolytic cleavage introduced
by radiolabeling of IGFBP-3, selected samples were reassayed using
unlabeled IGFBP-3 as a substrate and Western immunoblotting for
detection of proteolytic products. Samples were incubated with 50 ng
IGFBP-3 in a total volume of 50 µl 20 mM HEPES, pH 7.2,
with 1 mM CaCl2, 0.1% BSA for 1–5 h at 37 C.
The reaction were terminated by the addition of SDS sample buffer, and
the mixtures were processed by SDS-PAGE (12.5% or 15% gels),
electroblotted, and immunoblotted with an anti-IGFBP-3 antibody using
enhanced chemiluminescence technique as previously described (18).
Protease purification
PHS was pooled, and 10 ml was subjected to stepwise ammonium
sulphate precipitation. Preliminary results using increments of 5%
(vol/vol) 3.8 M ammonium sulfate, demonstrated that active
material precipitated between 30–50% (vol/vol) 3.8 M
ammonium sulfate. The 30–50% precipitate was resuspended in 20
mM Tris, pH 8.0, and dialyzed (10 kDa cutoff) overnight
against 20 liter 20 mM Tris, pH 8.0, at 4 C. The sample was
applied to a 6-ml Resource Q anion-exchange column (Pharmacia,
Stockholm, Sweden) developed with 20 mM Tris, pH 8.0.
Adsorbed proteins were eluted by a linear gradient of 0.0–1.0
M of NaCl. Active fractions, as determined by the IGFBP-3
protease assay, typically eluting at 0.08–0.15 M NaCl were
pooled. In preliminary experiments, contamination with IgG was present
at this step. To remove the IgG, the pooled fractions were adjusted to
pH 7.2 with 100 mM HEPES, pH 7.2, and run over a protein-A
Sepharose column (12 ml bed volume) developed with 50 mM
HEPES, pH 7.2. The IgG-free eluate was concentrated by Centricon 30
(Amicon, Danvers, MA) ultrafiltration and processed by size-exclusion
chromatography on a Superdex 200 HR column (Pharmacia) developed with a
50 mM phosphate buffer, pH 7.4, 150 mM NaCl.
Fractions were assayed for IGFBP-3 protease activity. Active fractions
were separated by SDS-PAGE (15% gels), electrotransfered to
polyvinylidene difluoride membranes and stained for total protein. A
band of approximately 70–90 kDa (on nonreducing gels) was then
subjected to N-terminal amino acid analysis as previously described
(21).
Characterization of IGFBP-3 proteases in PHS
To determine the molecular weight of the IGFBP-3 protease
activities in PHS and NHS, 50% polyethylene glycol 8000 was added to
1.5 ml of serum [final concentration 12% (wt/vol)] to precipitate
the proteolytic activity. The pellet was then solubilized in 1.5 ml 0.1
M NaHCO3 and concentrated to approximately 0.4
ml using a Centricon 50, and the buffer was exchanged by successive
addition of a total of 1.6 ml PBS, 0.01% Tween 80 and reconcentrated.
The samples were then applied to a Superdex 200 HR size-exclusion
column as described above. Fractions of 1 ml were collected, and
75-µl aliquots assayed for IGFBP-3 protease activity. The fractions
containing IGFBP-3 protease activity were divided into two pools based
on the molecular mass 70–90 kDa and >150 kDa. These two pools were
further characterized by incubation with 125I-IGFBP-3 with
or without various protease inhibitors and/or PAs. These samples were
analyzed as described in the IGFBP-3 protease assay.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). N-terminal amino acid analysis of
this material from a polyvinylidene difluoride membrane demonstrated a
sequence of EPLDDYVNTQG, which was consistent with the N-terminal
sequence of human plasminogen.
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150 and 70–90 kDa could be demonstrated (Fig. 2a). In contrast to PHS, NHS contained
only the IGFBP-3 protease activity of 70–90 kDa consistent with the
molecular mass of plasminogen (Fig. 2b). Pooled fractions from each of
the two activities from PHS were then incubated with
125I-IGFBP-3 in the absence or presence of protease
inhibitors and PAs, respectively. Figure 3 demonstrates that the >150-kDa pool
degraded 125I-IGFBP-3 into three major fragments of
approximately 29, 19, and 15 kDa, respectively. The 70- to 90-kDa pool
degraded 125I-IGFBP-3 into a slightly different pattern
with two major fragments of approximately 19 and 15 kDa and two minor
fragments of approximately 28 and 3 kDa, respectively. It is possible,
though, that the 3-kDa fragment results from further degradation of
larger fragments due to higher activity of the 70- to 90-kDa protease.
Addition of 5 mM EDTA or 0.5 mM
zinc2+ completely inhibited the activity of the >150-kDa
protease, but EDTA had no effect and zinc2+ only partially
inhibited the activity of the 70-to 90-kDa pool (Fig. 3). Concentrations as low as 0.1
mM zinc2+ completely inhibited the >150-kDa
protease activity (data not shown). Conversely, addition of 500 µg/ml
aprotinin or 20 µg/ml -2-antiplasmin had no effect on the activity
of the >150-kDa pool, whereas, these inhibitors completely blocked the
ability of the 70- to 90-kDa pool to proteolyze
125I-IGFBP-3. Another difference between the two protease
activities was their response to addition of tPA. Addition of 1 µg/ml
tPA to the >150-kDa pool (A + tPA) had no enhancing effect on its
activity, whereas, addition of tPA to the 70- to 90-kDa pool (B + tPA)
greatly enhanced its ability to proteolyze 125I-IGFBP-3
(Fig. 3). These data strongly suggest that PHS contains at least two
different molecular mass proteases capable of degrading 125
I-IGFBP-3, which have distinct inhibitor and activator profiles.
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, addition
of 1 µg/ml tPA, UK, or streptokinase to PHS slightly increased its
ability to degrade 125I-IGFBP-3. This increase is likely
due to activation of inactive plasminogen to plasmin. The degradation
pattern of PHS was also slightly altered by addition of PAs; this is
evidenced by the increase in intensity of a lower molecular mass band
of approximately 10 kDa. Addition of PAs to NHS greatly enhanced its
ability to degrade 125I-IGFBP-3 (Fig. 4). However, addition
of PAs to PDP had no significant effect on its ability to degrade
125I-IGFBP-3, consistent with the absence of
plasminogen. Some minor degradation bands were present in the PDP + tPA
sample, but these were likely due to the ability of tPA itself to
proteolyze IGFBP-3. This view was supported by the pattern of fragments
produced by addition of tPA to PDP, in particular with the two
fragments slightly above and below 30 kDa, similar to the fragmentation
pattern produced by tPA itself (see below).
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. In this experiment,
125I-IGFBP-3 was incubated with the >150-kDa pool (75
µl), tPA (10 µg/ml), and UK (10 µg/ml) in the presence or absence
of increasing concentrations of the PA inhibitor PPACK. As shown in
Fig. 5, all proteases were capable of degrading
125I-IGFBP-3. The proteolytic fragments produced by the
>150-kDa protease in PHS differed from those produced by tPA and UK,
with one band of approximately 30 kDa as opposed to two bands slightly
above and below 30 kDa, respectively. However, whereas both tPA and UK
were inhibited by PPACK, the >150-kDa protease was not. We also
incubated unfractionated PHS with PPACK and -2-antiplasmin at
concentrations that inhibit IGFBP-3 proteolysis by plasmin, tPA, UK,
and the 70- to 90-kDa protease. Neither PPACK nor -2-antiplasmin
decreased IGFBP-3 proteolytic activity of PHS (data not shown). NHS had
no significant activity without addition of PAs. These data indicate
that both tPA and UK can act as IGFBP-3 proteases alone, and that the
150-kDa protease activity does not appear to be related to the PAs
based on the inability of PPACK to alter its activity.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we demonstrated the presence of two proteases in
PHS that can degrade intact glycosylated 125I-IGFBP-3 into
smaller fragments. We have purified one of these protease activities
and demonstrated the major protein component to be identical with the
known fibrinolytic enzyme, plasminogen. Moreover, plasminogen IGFBP-3
proteolytic activity could also be demonstrated in NHS. In contrast to
NHS, the higher molecular mass (>150 kDa) protease activity was
present only in PHS. We propose that these two activities represent
distinct proteolytic activities based on the following findings. Size-
exclusion chromatography revealed that the two proteases are 70–90 kDa
and >150 kDa in size, respectively. The 70- to 90-kDa protease
activity is inhibited by the serine protease inhibitors aprotinin and
-2-antiplasmin, but is unaffected by EDTA. This was in contrast to
the >150-kDa protease activity, which demonstrated cation dependency,
because it was inhibited by EDTA and was unaffected by the above
protease inhibitors. Zinc completely inhibited the activity of the
>150-kDa protease, but only partly inhibited the 70- to 90-kDa
protease. Addition of PAs markedly increased the activity of the 70- to
90-kDa protease, but did not alter the activity of >150-kDa
protease.
Most nonpregnant serum and plasma samples lack the ability to proteolyze IGFBP-3 because of the presence of plasmin inhibitors and low levels of circulating free tPA. It is not clear how our purification protocol and/or size-exclusion chromatography activated the fibrinolytic system to degrade IGFBP-3 in vitro. It is possible that specific serum inhibitors of the fibrinolytic cascade were separated from the protease during the purification, or that the physical manipulation of the serum resulted in the activation of plasmin.
What is the physiological role of the fibrinolytic system in acting as an intravascular IGFBP-3 protease resulting in increased availability of circulating IGFs? Clearly, PAs can induce IGFBP-3 proteolytic activity in NHS, as has been described in this paper. Campbell et al. (22) previously showed that plasmin can act as an IGFBP-3 protease at the cellular level, where it was converted by osteosarcoma cells from endogenous plasminogen by UK or tPA residing at the cell membrane (25). Thus, increases in plasmin concentrations can enhance cell growth through the release of IGFs from IGFBPs (22, 26).
We also demonstrated that PAs, including tPA and UK, have IGFBP-3
proteolytic activity, and that they can act more potently by converting
endogenous serum plasminogen to plasmin. However, in vivo
inhibitors such as plasminogen activator inhibitor, -2-antiplasmin,
and -2-macroglobulin can act at each level of the proteolytic
cascade to prevent these proteolytic processes (27). The ubiquitous
presence of these inhibitors in serum likely prevents us from detecting
an overall increase in serum IGFBP-3 protease activity in situations
where local activation of the fibrinolytic system has taken place. As
further support, we were not able to measure increased arterial-venous
difference in IGFBP-3 protease activity in blood from exercising human
muscle, although local tPA expression was elevated (Bang, P., V. Berg,
B. Saltin, and K. Hall, unpublished observations). Other relevant
physiological actions of the plasmin system may include IGFBP-3
proteolysis and IGF release at local sites of tissue injury or blood
clot formation, thereby supporting mitogenesis of regenerating tissue.
In fact, the presence of a 30-kDa IGFBP-3 fragment, which constitutes a
significant proportion of circulating IGFBP-3 in normal healthy adults
(18), may suggest that a constant low level of intravascular
proteolysis of IGFBP-3 may occur in the absence of significant
circulating IGFBP-3 proteolytic activity. However, it cannot be
excluded that these IGFBP-3 fragments at least partially originate from
extravascular proteolysis (26).
In contrast to the fibrinolytic system, the distinct >150-kDa protease in term PHS is capable of exerting generalized IGFBP-3 proteolytic effects in serum. Whether this is due to a more widespread release of the protease in the circulation and/or less efficient inhibition in serum of an organ-specific activity is not known. Early mixing experiments have demonstrated that addition of NHS to PHS did not inhibit the IGFBP-3 protease (4, 6), which suggests that the latter case is true. If the protease activity is actually produced in the placenta, as it has been postulated, the protease that can be measured in serum may only be a fraction of the activity present in the maternal circulation at the maternal-placental interface.
The present study demonstrates a distinct >150-kDa protease activity that is unique to PHS, which could be completely inhibited by EDTA and zinc. Inhibition of matrix metalloproteinase (MMP)-1 and MMP-3 in unfractionated rat pregnancy serum, either by the addition of TIMP-1 or by specific antibodies to these MMPs, have demonstrated a marked reduction in the IGFBP-3 proteolytic activity (19). Therefore these MMPs have been suggested to be responsible for the IGFBP-3 proteolytic activity in rat pregnancy serum. Because the molecular mass of MMP-1 and MMP-3 in human serum is in the range of 50–60 kDa, these proteases are not likely to explain the activity of the >150-kDa protease in human pregnancy serum. In addition, MMPs are generally zinc dependent, but the >150-kDa protease was completely inhibited by zinc. We are currently performing experiments with human pregnancy serum using specific inhibitors of MMPs such as TIMP-1 and specific anti-MMP antibodies to find further support for this view. However, eventually isolation and characterization of the human protease is needed to fully resolve these questions.
It is well accepted that proteolysis of IGFBP-3 does not result in a disruption of the ternary complex between IGF, IGFBP-3, and ALS (5, 12, 18). However, Laserre et al. (7) demonstrated that IGF-I had a 10-fold and IGF-II a 2-fold decreased affinity for IGFBP-3 isolated from PHS compared with NHS. When compared with NHS, IGF-I and -II dissociated 10 and 6 times faster from IGFBP-3 in PHS, respectively, and the proportion of IGF-I in the free form was nearly 3 times greater in PHS than in NHS. These data suggest that although in PHS most IGF-I and -II is still present in the 150-kDa complex, its bioavailability is likely increased.
Thus, the presence of at least two distinct proteases in human serum may prove to play an important role in the regulation of the endocrine actions of IGF-I. The challenge now is to determine the identity of the pregnancy-specific protease activity and possible other proteases, as well as to study their regulation in relation to physiological and pathophysiological states in humans.
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Acknowledgments |
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Footnotes |
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1 This work was supported by grants from the Swedish Medical Research
council (B96–19X-11634–01A), the Foundation for Studies Without
Animals, Swedish Foundation for Childrens Ward. It was presented in
part at the 3rd International Meeting on Insulin-Like Growth Factor
Binding Proteins, Tübingen, Germany, October, 1995 and in the
proceedings from this meeting: Bang P. 1995 Serum proteolysis of
IGFBP-3. Prog Growth Factor Res 6:285–292. 
Received March 13, 1997.
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