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(Received for publication, July 15, 1994; and in revised form, December 12, 1994)
From the
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
sample buffer
containing dithiothreitol and heated for 10 min at 60 °C. The
entire volume was placed in a single well using a 5-slot comb. After
electrophoresis for 1 h at 25 mA, the current was increased to 50 mA,
and electrophoresis was terminated after another 2.5 h. The proteins
were transferred to an Applied Biosystems ProBlott polyvinylidene
difluoride membrane and localized with Coomassie Blue. The 14- and
18-kDa bands were excised with a razor blade and subjected to Edman
degradation using an Applied Biosystems Model 473A sequencer equipped
with a BLOTT cartridge.
cartridges (Waters Associates). The PCR
mutagenesis primers were 45-mer oligonucleotides with the following
sequences: M1, 5`CTCCCGGGGCGCCCCATTGACCTTCATCTGGCCCCCACTGGT-3`; M2,
5`-CTCCCGGGGCGCCCCATTGACCTGCATCTTGCCCCCACTGGT-3`; M3,
5`-CTCCCGGGGCGCCCCATTGACCTTCAGCTTGCCCCCACTGGT-3`; and M4,
5`-CTCCCGGGGCGCCCCATTGACCTTCTCCTTGCCCCCACTGGT-3`. The NarI-containing synthetic DNA fragment for pBluescript
SK
consisted of two oligonucleotides: RL1-27
(5`-AATTCGATATCAAGCTTGGCGCCACCG-3`) and RL2-27
(5`-TCGACGGTGGCGCCAAGCTTGATATCG-3`).
(Stratagene, La
Jolla, CA) with EcoRI and SalI and replacing the
small multiple cloning site segment (containing the ClaI site)
with a synthetic double-stranded DNA fragment containing a NarI site flanked by EcoRI and SalI sites.
Mutations were generated in IGFBP-4 cDNA by PCR using mismatched
primers (M1-M4). PCR was performed according to the suppliers of
the PCR kit (Perkin-Elmer). Thirty cycles of PCR were performed in a
Perkin-Elmer DNA thermal cycler with each cycle consisting of a 1-min
denaturation step at 94 °C, a 2-min annealing step at 55 °C,
and a 3-min extension step at 72 °C. An additional 7-min extension
step was included after the last cycle. The 5`-PCR primer and the
IGFBP-4 cDNA used as template were from the pBS24Ub-IGFBP-4
construction(5) . The 3`-reverse PCR primers (M1-M4) were
complementary to the nucleotide sequence encoding amino acids
129-143 of mature IGFBP-4, but contained mismatches at
appropriate codons to generate the mutants shown in Table 2. The
M3 and M4 PCRs also contained 2.5% formamide(5) . The PCR
products were treated as described (5) and then digested with SstII and NarI (located within the nucleotide
sequences encoding amino acids 139-141), gel-purified, and
ligated to SstII/NarI-digested
pBlsc-BP4(140-237) to generate full-length IGFBP-4 mutants. These
constructs were sequenced to confirm the mutations. The full-length
IGFBP-4 mutant cDNAs were then excised with SstII and SalI, gel-purified, ligated into the yeast expression vector
pBS24Ub, and introduced into Escherichia coli strain HB101 as
described(5) .
column (Macherey Nagel,
Düren, Germany). Fractions were analyzed by
SDS-PAGE with Western ligand blotting and silver staining. The IGFBP-4
fractions that appeared pure by these analyses were pooled, and the
protein content was determined by the method of Lowry et al.(28) and by weighing the lyophilized material.
I-IGF overnight at 4 °C, and visualized
by autoradiography according to the method of Hossenlopp et al.(13) and as described
previously(7, 9, 10) . Unstained molecular
weight standards (Bio-Rad) were processed in parallel, and proteins
were stained using India ink(14) . Films were scanned with an
UltroScan XL laser densitometer; absorbance curves were integrated, and
molecular size was determined using GelScan XL software (Pharmacia
Biotech Inc.).
I-IGF (30,000 cpm, 0.02 nM) and various
concentrations of unlabeled IGF overnight at 4 °C. (The
concentration of IGFBP-4 was based on a preliminary titration
experiment to determine a concentration near but not at the plateau of
maximal radioligand binding.) 1% activated charcoal containing 0.2
mg/ml protamine sulfate was added, and the samples were centrifuged at
4 °C to separate bound from free IGF-I(7, 15) . A
control value for binding in buffer alone (nonspecific binding) was
subtracted from the total bound radioactivity to determine a specific
binding value. Nonspecific binding ranged from 3 to 10% of total counts
added.
H]Aminoisobutyric acid (AIB)
uptake was determined as described previously(7) . Confluent
bovine fibroblasts were washed three times with Hanks' balanced
saline solution containing 1.75 g/liter NaHCO, 20 mM Hepes (pH 7.4), and 0.1% bovine serum albumin. The medium was
changed to that containing IGF-I or insulin with and without rhIGFBP-4
or mutant IGFBP-4, and the monolayers were incubated at 37 °C for 6
h. [H]AIB (0.5 µCi/ml, 8 µM) was
added, and incubation was continued for 12 min. Cultures were placed on
ice, and cells were washed quickly four times with cold
phosphate-buffered saline. Monolayers were solubilized in 0.25 N NaOH, and aliquots were taken for liquid scintillation counting.
Results are expressed as the percentage of total counts in the
incubation medium that were taken up by the cells.
H]Thymidine incorporation
was determined as described previously(16) . Confluent human
fibroblast cultures were washed twice with a 1:1 mixture (v/v) of
Waymouth medium/Dulbecco's modified Eagle's medium plus
0.1% bovine serum albumin, preincubated in this serum-free medium (SFM)
for 6 h, washed again, and exposed to SFM for 40 h. IGF-II or an
equivalent volume of SFM, with and without rhIGFBP-4 or mutant IGFBP-4,
was added to this medium for 5 h. IGF-I or insulin was then added to
the conditioned medium, and [H]thymidine
incorporation was measured at 22-26 h. Results are expressed as
the percentage of total counts in the incubation medium that were
incorporated into acid-precipitable material.
-terminal amino acid
sequences shown in Table 1. The 18-kDa IGFBP-4 fragment
corresponded to the NH-terminal half of the protein. The 15
NH-terminal amino acids are identical to those of native
IGFBP-4 (where X = Cys), except for an additional
NH-terminal Arg that occurs in 90% of rhIGFBP-4, as
previously noted(5) . The 14-kDa IGFBP-4 fragment corresponded
to the COOH-terminal half of the mature IGFBP-4 molecule. The
NH-terminal amino acid of the 14-kDa fragment suggests
cleavage after methionine at position 135 of the mature protein.
Incubation of rhIGFBP-4 either in human fibroblast or human
osteoblast-like cell conditioned medium generated fragments with
identical sequences.
24 kDa) when analyzed by nonreducing SDS-PAGE (Fig. 1). In addition, the mutants displayed an HPLC elution
profile similar to that of rhIGFBP-4(5) .
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.
H]thymidine incorporation 3-fold. The addition
of wild-type IGFBP-4 with IGF-II did not alter this augmentative
effect. However, the addition of M1, M2, or M4 significantly inhibited
IGF-II-enhanced IGF-I stimulation. These IGFBP-4 mutants had no effect
alone and did not influence insulin-stimulated
[H]thymidine incorporation. Similar results were
obtained with M3 in a separate experiment (data not shown). As noted in Table 4, in the absence of proteolysis, wild-type and mutant
IGFBPs-4 equivalently inhibited IGF-I-stimulated
[H]thymidine incorporation.
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.
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