(Received for publication, July 15, 1994; and in revised form, December 12, 1994)
From the
Cultured human fibroblasts and osteoblast-like cells secrete an
insulin-like growth factor (IGF)-dependent protease that cleaves
IGF-binding protein-4 (IGFBP-4) into two fragments of 18 and 14
kDa. Edman degradation of the isolated proteins established the amino
termini of the reaction products. Sequence analysis of the 14-kDa
carboxyl-terminal half of IGFBP-4 suggested cleavage after methionine
at position 135 of the mature protein. Four variant IGFBP-4 molecules
with single amino acid substitutions around this cleavage site were
constructed and expressed. Wild-type and mutant IGFBPs-4 bound IGF-I
and IGF-II with equivalent affinities and, in the intact state, were
equally effective inhibitors of IGF-I action. However, the IGFBP-4
mutants were relatively resistant to IGF-dependent proteolysis. A
5-6-h incubation in human fibroblast conditioned medium in the
presence of IGF-II was sufficient for near total hydrolysis of
wild-type IGFBP-4, whereas the mutant IGFBPs-4 were only minimally
affected at this time. After a 24-h incubation with IGF-II, all mutant
IGFBPs-4 showed extensive proteolysis, generating 18- and 14-kDa
fragments. Pre-exposure of human fibroblasts in serum-free conditioned
medium to IGF-II for 5 h potentiated subsequent IGF-I stimulation of
DNA synthesis. When added with IGF-II, the protease-resistant mutant
IGFBPs-4, but not wild-type IGFBP-4, suppressed IGF-II enhancement of
IGF-I-stimulated DNA synthesis. These biological studies suggest that
the IGFBP-4/IGFBP-4 protease system may play a role modulating local
cellular response to IGF-I.
Insulin-like growth factor-binding protein-4 (IGFBP-4) ()is expressed and secreted by a variety of cell types and
is an effective inhibitor of IGF action in vivo and in
vitro(1, 2, 3, 4, 5, 6, 7) .
Regulation of IGFBP-4 bioavailability occurs at the level of IGFBP-4
gene expression and also through post-translational modification of the
secreted protein. We (7, 9, 10) and others (8, 11) have identified an IGF-dependent IGFBP-4
protease secreted by human fibroblasts and human osteoblast-like cells
that cleaves the IGFBP-4 molecule (24 kDa unreduced, 32 kDa reduced)
into two fragments of
18 and 14 kDa. This specific proteolytic
cleavage decreases the affinity of IGFBP-4 for IGF peptide, resulting
in increased IGF bioactivity(7) . Thus, the IGFBP-4/IGFBP-4
protease system, by virtue of its tight and focused control of IGF
action, could be important in regulating localized cell growth.
In this study, we identify an IGFBP-4 cleavage site by sequencing purified 18- and 14-kDa reaction products of IGF-dependent IGFBP-4 proteolysis. Using this information, we constructed and expressed variant IGFBP-4 molecules with amino acid substitutions around the cleavage site. These ``proteolysis'' mutants were employed to study the role of the IGFBP-4/IGFBP-4 protease system in modulating cellular response to IGFs.
Figure 1: SDS-PAGE analysis of wild-type and mutant rhIGFBPs-4. 300 ng of HPLC-purified rhIGFBP-4 (lanea), M1 (laneb), M2 (lanec), M3 (laned), and M4 (lane e) were fractionated on a 7.5-15% acrylamide gel under nonreducing conditions, and the gel was silver-stained. Migration positions of molecular size markers (in kilodaltons) are shown on the left.
All four IGFBP-4
mutants were able to bind radiolabeled IGF-I and IGF-II on Western
ligand blotting. Furthermore, the affinity for IGF-I and IGF-II was not
appreciably altered by the mutations. IGFs were equipotent in competing
for radiolabeled IGF-I and IGF-II binding to ``wild-type''
and mutant IGFBPs-4 (Fig. 2). 50% displacement of I-IGF-I and
I-IGF-II from each IGFBP-4
mutant was seen with unlabeled IGF at
0.05 and 0.06 nM,
respectively. Scatchard analysis of the data from three experiments
estimated an equilibrium constant of
2
10
M
for rhIGFBP-4, which agrees with
our earlier study(5) . Equilibrium constants for the IGFBP-4
mutants did not differ significantly from that for rhIGFBP-4 (Table 3).
Figure 2:
Competitive inhibition of I-IGF-I and
I-IGF-II binding to IGFBP-4
mutants. Various concentrations of unlabeled IGF-I or IGF-II were added
to compete for
I-IGF-I binding (leftpanel) and
IIGF-II binding (rightpanel) to M1 (
), M2 (&cjs3570;), M3 (
), M4
(
), and wild-type rhIGFBP-4 (
) as described under
``Experimental Procedures.'' Results are means of three
determinations expressed as percent of maximum specific
I-IGF-I binding (26, 29, 22, 32, and 18%) or
I-IGF-II binding (33, 31, 30, 38, and 35%) for wild-type
rhIGFBP-4 and M1-M4, respectively.
The IGFBP-4 mutants were potent inhibitors of
IGF-I-stimulated [H]AIB uptake in cultured bovine
fibroblasts. Bovine fibroblasts are exquisitely responsive to IGF-I and
do not degrade IGFBP-4 during the bioassay; therefore, this system can
be used to evaluate function of the intact IGFBP-4
molecule(7) . As indicated in Fig. 3, the presence of 10
nM wild-type IGFBP-4, M1, M2, M3, or M4 completely inhibited
the
7-fold increase in [
H]AIB uptake
stimulated by 2 nM IGF-I. Half-maximal effectiveness was seen
with
4 nM mutant and wild-type IGFBPs-4 (data not
shown)(7) . Exogenous wild-type and mutant IGFBPs-4 had no
effect alone and did not influence insulin-stimulated
[
H]AIB uptake in these cells. When added with
IGF-I, a 5-fold molar excess of wild-type and mutant IGFBPs-4 inhibited
IGF-I stimulation of [
H]thymidine incorporation
in human fibroblasts by
70% (Table 4).
Figure 3:
Effect of mutant IGFBP-4 on IGF-I- and
insulin-stimulated [H]AIB uptake in bovine
fibroblasts. Bovine fibroblasts were washed and incubated for 6 h with
2 nM IGF-I or 100 nM insulin with or without the
indicated IGFBP-4 at 10 nM. [
H]AIB
uptake was measured as described under ``Experimental
Procedures.'' Results are means ± S.E. of three
determinations. The asterisks indicate a significant effect of
IGFBP-4 (p < 0.05)
Figure 4: Cell-free IGFBP-4 protease assay. 50 ng of wild-type rhIGFBP-4 and mutant IGFBP-4 (M1 and M2) were incubated at 37 °C in human fibroblast conditioned medium under cell-free conditions without(-) or with (+) 5 nM IGF-II for the indicated times. Samples were analyzed by Western ligand blotting. The arrow indicates the migration position of 24-kDa IGFBP-4.
Figure 5:
Time course for cell-free IGF-dependent
IGFBP-4 proteolysis. Proteolysis of wild-type rhIGFBP-4 (), M1
(
), M2 (&cjs3570;), M3 (
), and M4 (
) with time was
determined as described in the legend to Fig. 4. Results are
expressed as percent of intact IGFBP-4 at t = 0. Each
point represents the mean value of three separate
experiments.
Figure 6: Immunoblot analysis using IGFBP-4 antiserum. Wild-type and mutant IGFBPs-4 (100 ng) were incubated for 24 h in human fibroblast conditioned medium under cell-free conditions without(-) or with (+) 5 nM IGF-II. Reduced samples were electrophoresed and transferred to nitrocellulose, and the filter was incubated with antiserum to IGFBP-4 (1:500 dilution) as described under ``Experimental Procedures.'' Migration positions of unstained molecular size marker (in kilodaltons) are shown on the left. Arrows indicate 18- and 14-kDa IGFBP-4 fragments.
Figure 7:
IGF-II-enhanced, IGF-I-stimulated
[H]thymidine incorporation in human fibroblasts:
effect of IGFBP-4 mutants. Human fibroblasts were washed and changed to
SFM for 40 h. 4 nM IGF-II (shaded and dotted
bars) or an equivalent amount of SFM (solidbars), with or without the indicated IGFBP-4 (25
nM), was added to the medium, and incubation was continued for
5 h. IGF-I (5 nM) or SFM (control (C)) was then
added, and [
H]thymidine incorporation was
measured at 22-26 h as described under ``Experimental
Procedures.'' Results are means ± S.E. of three
determinations. The asterisks indicate a significant effect of
IGFBP-4 (p < 0.05).
These data demonstrate that the IGF-dependent IGFBP-4
protease secreted by human fibroblasts and human osteoblast-like cells
cleaves the IGFBP-4 molecule at a single site on the carboxyl-terminal
side of methionine 135, producing fragments of 18 and 14 kDa.
Furthermore, studies with IGFBP-4 mutated at this cleavage site
indicate that inhibition of this proteolytic processing of IGFBP-4 can
have biological consequences.
Sequence analysis of the 14-kDa
proteolysis product predicted a precise cleavage event between
methionine and lysine of IGFBP-4
(Met-Lys
). Tissue kallikreins are
known to cleave peptide bonds between methionine and lysine and between
arginine and serine in kininogen to release
lysylbradykinin(17) . Thus, these results suggest that
IGF-dependent IGFBP-4 cleavage may be the product of the activity of a
hydrolytic enzyme with a specificity similar to that of the
kallikreins. Preliminary data of Chernausek et al.(18) also suggest that the IGFBP-4 protease secreted by
B104 rat neuroblastoma cells may be a kallikrein-like enzyme. However,
IGF-dependent proteolysis of IGFBP-4 in human fibroblast or
osteoblast-like cell conditioned medium was not inhibited by
conventional trypsin- or chymotrypsin-like serine protease inhibitors.
EDTA and 1,10-phenanthroline were by far the most effective inhibitors (7, 9) , and proteolytic activity could be restored
with calcium in EDTA-treated samples and with zinc in
1,10-phenanthroline-treated samples. (
)These protease
inhibitor results may indicate a novel calcium-dependent
metalloprotease with specificity similar to that of kallikreins.
Alternatively, it is possible that the specific cleavage occurs several
residues upstream of methionine 135 and that the 14-kDa fragment is
further processed by the action of an aminopeptidase. COOH-terminal
analysis of the 18-kDa IGFBP-4 fragment may help establish the
involvement of additional enzymes. Similar immunoreactive fragments (Fig. 6) and the fact that sequencing of the 18- and 14-kDa
products generated from IGF-dependent proteolysis of the four IGFBP-4
mutants yielded the same amino termini as wild-type rhIGFBP-4 (
)argue against a secondary cleavage site being utilized as
a result of a block in the mutated site. Regardless, our results
clearly show that the cleavage site is located in domain 2 of IGFBP-4,
which is the central nonconserved region of the
IGFBPs(19, 20, 21, 22) . The
conserved domains (domains 1 and 3) or NH
- and
COOH-terminal portions of the IGFBPs are known to be important for IGF
binding(23, 24) , leaving the possibility that the
nonconserved domain 2 could be involved in regulating the activity
and/or tissue specificity of each IGFBP. Our observations that IGFBPs-4
mutated in domain 2 have binding affinities similar to those of
wild-type IGFBP-4 yet are partially resistant to proteolysis support
this concept.
Biological studies using IGFBPs-4 with single amino acid mutations around the predicted cleavage site indicated that we were at or near the correct site. The expressed wild-type and mutant IGFBPs-4 bound IGF-I and IGF-II with equivalent affinities and were effective inhibitors of IGF-I action when assessed for function of the intact IGFBP-4 molecule. One way in which the mutant IGFBPs-4 differed from wild-type IGFBP-4 was in their relative resistance to IGF-dependent proteolysis. A 5-6-h incubation in human fibroblast conditioned medium in the presence of IGF-II was sufficient for near total hydrolysis of wild-type IGFBP-4, whereas the mutant IGFBPs-4 were only minimally affected.
As demonstrated in the assays for function of intact IGFBP-4, the IGFBP-4 mutants were not super-inhibitors of IGF action. However, resistance to proteolysis corresponded with increased effectiveness of mutant IGFBP-4 as a physiological inhibitor of cellular IGF-I action. Pre-exposure of human fibroblasts to IGF-II results in marked enhancement of subsequent IGF-I-stimulated DNA synthesis and cell replication via effects on pericellular IGFBPs(16) . The addition of wild-type rhIGFBP-4, which is rapidly degraded in the human fibroblast system(7) , did not affect IGF-II potentiation of IGF-I action. The finding in the present study that protease-resistant IGFBP-4 mutants suppressed this effect indicates that IGFBP-4 proteolysis contributes to IGF-II enhancement of IGF-I action. In addition, functional interplay between other IGFBPs and local agents or distinct biological effects of 18- and/or 14-kDa IGFBP-4 fragments are likely to be important and were not addressed in this study. The latter possibility is particularly intriguing since IGFBP-3 and IGFBP-5 fragments generated through proteolytic processing appear to be stimulatory, whereas the intact forms of IGFBP are clearly inhibitory(25, 26, 27) . Future studies are aimed at acquiring a better understanding of the IGFBP-4/IGFBP-4 proteolytic system and its role in the control of cell growth.