MDCK cells secrete neutral proteases cleaving
insulin-like growth factor-binding protein-2 to -6
Liliana
Shalamanova,
Bernd
Kübler,
Jens-Gerd
Scharf, and
Thomas
Braulke
University of Hamburg, Children's Hospital, Department of
Biochemistry, D-20246 Hamburg; and Division of Gastroenterology and
Endocrinology, Department of Medicine, University of
Göttingen, D-37075 Göttingen, Germany
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ABSTRACT |
Proteolysis of
insulin-like growth factor-binding proteins (IGFBPs) may be an
important mechanism to regulate IGF availability and IGF-independent
functions of IGFBPs. We analyzed the secretion of IGFBP proteases in
Madin-Darby canine kidney (MDCK) cells. The results showed that several
specific proteases were secreted, cleaving IGFBP-2 to -6 at neutral pH.
The proteolytic activity against IGFBP-6 differed at least from IGFBP-5
protease activity in its sensitivity both to IGF-II and to the
hydroxamic acid-based disintegrin metalloprotease inhibitor, as well as
serine protease inhibitors. During partial purification steps, the
serine protease inhibitor-sensitive fraction with IGFBP-6 protease
activity was separated from fractions characterized by the presence of
a 30-kDa disintegrin immunoreactive band. Whereas the IGFBP-4 and -6 proteases are predominantly secreted across the basolateral membrane,
the majority of IGFBPs are sorted to the apical medium from
filter-grown cells. These studies indicate that the side-specific
secretion of several distinct IGFBP proteases with partially
overlapping IGFBP specificities may be another level in the regulation
of IGF-dependent epithelial functions.
insulin-like growth factor-binding proteins and proteases; polarized sorting; disintegrin metalloprotease
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INTRODUCTION |
INSULIN-LIKE GROWTH
FACTORS (IGFs) I and II are single-chain polypeptides expressed
in many tissues and participate in the regulation of growth and
differentiation of various cell types as autocrine and/or paracrine
factors. Most of the metabolic and mitogenic effects of IGFs are
mediated by IGF-I or insulin receptors exhibiting tyrosine kinase
activity upon ligand binding (18). The capacity of IGFs to
affect cell growth and metabolism via interaction with cell surface
receptors is controlled by a family of IGF-binding proteins
(IGFBPs). Six distinct high-affinity IGFBPs, designated IGFBP-1 to
IGFBP-6, have been characterized, differing in molecular mass,
posttranslational modifications, and tissue and developmental
regulated expression (10, 44). Additional components of
the IGF system are IGFBP-specific proteases. Limited proteolysis of
IGFBPs is believed to be the major mechanism for the release of IGFs
from IGFBP · IGF complexes, generating fragments with reduced
affinity for IGFs (26). Proteolytic activity at neutral pH
has been detected for IGFBP-2 (15), IGFBP-3 (16, 42), IGFBP-4 (14, 22, 30), and IGFBP-5
(9, 39), which are characterized by distinct fragmentation
patterns, substrate specificity, and inhibitor profile. Furthermore,
several studies suggest that acidic proteases may be involved in
inactivation and the regulation of the extracellular IGFBP level,
presumably in the lysosomal degradation pathway (2, 3, 8, 12, 27,
36). Two IGFBP proteases, the IGF-dependent IGFBP-4 protease and
the IGFBP-5 serine protease secreted by cultured fibroblasts, have
recently been reported to be identical with the pregnancy-associated plasma protein A and the complement component C1s, respectively (6, 23).
IGFs, IGFBPs, and IGF receptors have been reported to
contribute to processes of cell proliferation and differentiation of epithelial cells (11, 32, 46). Prerequisite for the
constitution of an epithelial permeability barrier are highly polarized
cells characterized by morphologically, functionally, and biochemically distinct apical and basolateral plasma membranes. The basolateral localization and secretion of IGF receptors and IGF-II, respectively, as well as differential sorting of IGFBP-2, -4, and -6 in polarized HT29-D4 colon carcinoma cells (33), demonstrate the
complexity of IGF-mediated epithelial cell regulation. Because
nothing is known about the synthesis of IGFBP proteases in
epithelial cells, the Madin-Darby canine kidney (MDCK) cell line was
used as a model to examine and characterize secreted IGFBP proteases.
Here we report that MDCK cells secrete different proteases cleaving
IGFBP-2 to -6. Proteases cleaving IGFBP-4 and -6 are delivered
preferentially to the basolateral side.
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MATERIALS AND METHODS |
Materials.
Sodium [125I]iodine (carrier free; specific activity 16.9 mCi/µg iodine), [35S]methionine (100 Ci/mmol), and
prestained protein standard (Rainbow) were purchased from
AmershamPharmaciaBiotech (Freiburg, Germany). Recombinant human
(rh)IGF-II from GroPep (Adelaide, Australia) was iodinated by the
chloramine T method (45) to a specific activity of
60-80 µCi/µg. Recombinant IGFBP-1 and IGFBP-2 and -5 were
purchased from UBI (Lake Placid, NY) and GroPep, respectively. Nonglycosylated rhIGFBP-3 was a generous gift from Drs. A. Sommer and
C. Maack (Celtrix, Santa Clara, CA). rhIGFBP-4 and -6 produced in yeast
(20) were kindly provided by Dr. J. Zapf (University Hospital, Zurich, Switzerland). Recombinant human arylsulfatase A (ASA)
was a kind gift from Dr. T. Dierks (University of Göttingen, Germany). The IGFBPs and ASA were iodinated using IODO-GEN (Pierce Chemical, Rockford, IL) as described (31). The hydroxamic
acid-based metalloprotease inhibitor TAPI was prepared at Immunex
(Seattle, WA) (29) and was a kind gift from Dr. S. Rose-John (University of Kiel, Kiel, Germany). Antibodies directed
against the prodomain (rb 132), the cystein-rich domain (rb 122), and
the disintegrin domain (rb 119) of human ADAM 12 S (25)
were kindly provided by Dr. U. Wewer (University of Copenhagen,
Copenhagen, Denmark). Peroxidase-conjugated goat anti-rabbit IgG came
from Dianova (Hamburg, Germany).
Blot analysis.
125I-labeled IGF-II ligand blot analysis was performed
according to Hossenlopp et al. (16), with slight
modifications. Briefly, conditioned media of 0.35 ml were mixed with
0.82 ml of ice-cold 96% ethanol and kept on ice for 1 h.
Precipitated probes were solubilized and separated by SDS-PAGE (12.5%
acrylamide) under nonreducing conditions. After electrotransfer and
blocking in 1% fish gelatin, the nitrocellulose membranes were probed
with 125I-IGF-II and exposed to X-ray films (X-Omat AR,
Kodak). For immunoblotting, the membranes were blocked with 5% nonfat
skim milk and probed with the rb 119, 122, and 132 antisera (dilution
1:100-1:500) and anti-rabbit IgG coupled to horseradish peroxidase
(dilution 1:15,000). Reactive bands were visualized by the SuperSignal
enhanced chemiluminescence detection system (Pierce Chemical) and
exposure to X-ray films.
Cell culture.
MDCK cells were maintained in Dulbecco's modified Eagle's medium
(DMEM) supplemented with 10% (vol/vol) fetal calf serum (FCS), 2 mM
glutamine, 4.5 g/l (wt/vol) glucose, and penicillin/streptomycin. The
MDCK cells were routinely grown on 35-mm dishes (Greiner, Germany) or,
to obtain polarized cell monolayers, on 24-mm Transwell (Costar,
Cambridge, MA) (0.4-µm pore size) polycarbonate filters, as described
recently (5). Serum-free medium conditioned for 24-72
h in the presence or absence of IGF-II (50 nM) was obtained as
described earlier (4).
Northern blot analysis.
The isolation of total RNA from MDCK cells, radiolabeling by random
priming of cDNA probes for human IGFBPs, and the Northern blot
hybridization were carried out as described previously
(35).
IGFBP protease assay.
Fifty microliters of conditioned media or 5-20 µl of column
fractions dialyzed against 20 mM Tris · HCl, pH 7.4, containing 10 mM NaCl, were incubated with 125I-labeled IGFBPs
(5,000-10,000 cpm) at 37°C for 6-18 h. When indicated, protein inhibitors were included. After solubilization, the samples were subjected to SDS-PAGE (12.5% acrylamide) and visualized by autoradiography, as described previously (24), or by
phosphorimaging (Cyclone, Packard, Meriden, CT).
Protease purification.
Three hundred milliliters of conditioned MDCK medium were sequentially
precipitated with 30 and 45% ammonium sulfate. The latter precipitate
was dissolved in 3 ml 20 mM Tris buffer, pH 7.4, containing 10 mM NaCl
(TBS) and was desalted by Sephadex G25 column chromatography
(PharmaciaBiotech, 1.0 × 35 cm). Two-milliliter fractions were
collected at a flow rate of 0.8 ml/min, and 50-µl aliquots were
assayed for IGFBP-6 protease activity. Active fractions were pooled and
loaded onto a 2-ml DEAE-Sephadex column equilibrated with TBS. The
column was washed with the same buffer (1 ml/min) until absorbance (280 nm) returned to the baseline. The proteins were eluted with a stepwise
gradient of 0.1, 0.25, 0.5, 0.75, and 1.0 M NaCl in 20 mM Tris buffer,
pH 7.5. One-milliliter fractions were collected, dialyzed against TBS,
and tested for IGFBP-6 protease activity. The protease-containing
fractions were pooled (fractions 7 and 8 to
pool I, fractions 9-12 to pool
II, and fractions 13-15 to pool III),
dialyzed against 50 mM Tris buffer, pH 7.4, and loaded onto a
hydroxyapatite column (PharmaciaBiotech; 1.0 × 1.0 cm). After the
column had been washed, the proteins were eluted with a two-step
gradient of 150 and 300 mM KPi in 50 mM Tris buffer, pH
7.4.
Other methods.
Hexosaminidase activity was determined as described earlier
(40). To examine the secretion of total newly synthesized
proteins, cells were labeled with [35S]methionine (80 µCi/ml) for 4.5 h. Aliquots (20%) of the apical or basolateral
media were precipitated with 1 ml of ice-cold acetone at
20°C for
24 h. After centrifugation, the pellets were dried and solubilized
for SDS-PAGE under reducing conditions. In parallel, aliquots of media
were used to determine the total radioactivity incorporated in secreted
proteins by trichloroacetic acid precipitation. The radiolabeled
polypeptides visualized by autoradiography or phosphorimaging were
quantified by densitometric scanning (Hewlett-Packard Scan Jet 4c/T and
the Advance Image Data Analyzer programme, Raytest, Straubenhardt, Germany).
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RESULTS |
Secretion of IGFBP proteases.
When conditioned media from MDCK cells were tested for IGFBP protease
activity at neutral pH using 125I-labeled rhIGFBP-1 to -6 as substrates, no fragmentation of IGFBP-1 was detected within a 6-h
incubation period. During the cell-free incubation, the
125I-IGFBP-2 was cleaved into two fragments of 24 and 13.5 kDa, and the 30-kDa nonglycosylated 125I-IGFBP-3 was
cleaved into four fragments of 25, 21, 15, and ~8.5 kDa (Fig.
1). 125I-IGFBP-4 was
fragmented to 17- and 10-kDa products, and the 125I-IGFBP-5
was cleaved to a 22-kDa doublet, 16- and 8-kDa peptide fragments, in
the presence of conditioned MDCK medium. 125I-IGFBP-6 was
almost completely hydrolyzed, depending on the incubation time, with a
transient appearance of 17-, 10-, and 6-kDa IGFBP-6 fragments (Fig. 1).
When the unrelated 62-kDa 125I-ASA was incubated with
conditioned MDCK medium for 14 h at 37°C under conditions
identical to 125I-IGFBP-3, no ASA proteolysis products were
detected (not shown).

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Fig. 1.
Proteolysis of insulin-like growth factor-binding
proteins (IGFBPs) by conditioned media from Madin-Darby canine kidney
(MDCK) cells. 125I-labeled IGFBP-1 to -6 were incubated
with 50 µl of nonconditioned (1) or 72-h-conditioned
media from MDCK cells (2) at 37°C and pH 7.4 for 6 h (IGFBP-1 and -5) or 12 h (IGFBP-2, -3, -4, and -6). Samples were
analyzed by SDS-PAGE and autoradiography. Experiments were carried out
1-2 times with a single medium of 3 different MDCK cell
cultures.
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Characterization of IGFBP proteases secreted by
MDCK cells.
To characterize the IGFBP protease(s) present in media of MDCK cells in
more detail, the experiments were carried out using 125I-IGFBP-4, -5, or -6 as representative substrates, as
indicated. The pH optimum of proteolytic activities with
125I-IGFBP-4 and -6 as substrates was estimated
between 6.5 and 7.4 (not shown). By the addition of 50 nM IGF-II to the
conditioned medium from MDCK cells, neither the proteolysis of
125I-IGFBP-4 nor that of 125I- IGFBP-5 was
affected, whereas the protease activity cleaving 125I-IGFBP-6 was efficiently inhibited in vitro under these
conditions (Fig. 2). Time course
experiments, however, showed that the final degradation of IGFBP-6 was
not prevented by IGF-II, whereas the rate of degradation was prolonged
(not shown). To classify the proteases catalyzing the cleavage of the
IGFBPs according to their inhibitor profile, media from MDCK cells were
incubated with 125I-IGFBP-5 or -6 in the presence or
absence of the metalloprotease inhibitors 1,10-phenanthroline
(Phe, 10 mM) and TAPI (0.1 mM) and the serine protease inhibitors
3,4-dichloroisocoumarin (0.2 mM) and aprotinin (Apr, 0.3 µM). The
proteolysis of 125I-IGFBP-5 was hardly affected by the
inhibitors tested with the exception of the formation of the 8-kDa
IGFBP-5 fragment, which was inhibited by TAPI and aprotinin (Fig.
3). In contrast,
125I- IGFBP-6 proteolytic activity in conditioned medium
was almost completely inhibited by Phe and TAPI with the exception of
the formation of 4-8% of the 17-kDa fragment, whereas the serine
protease inhibitors impaired the proteolysis of
125I-IGFBP-6 moderately or weakly (not shown for Apr).
These data suggest that 1) different IGF-sensitive IGFBP
proteases are present in the medium of MDCK cells and 2)
more than one protease with a distinct inhibitor profile may be
involved in the degradation of a specific IGFBP.

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Fig. 2.
Effect of insulin-like growth factor II (IGF-II) on the
proteolysis of IGFBP-4, -5, and -6 in conditioned media from MDCK
cells. 125I-labeled IGFBP-4, -5 and -6 were incubated with
50 µl of nonconditioned (Co) or conditioned media (72 h) for 8 h
at 37°C at neutral pH. Aliquots of conditioned media were incubated
with 125I-IGFBPs in the presence (+) or absence ( ) of 50 nM IGF-II, followed by SDS-PAGE and autoradiography. Experiments were
carried out 1-2 times with a single medium of 3 different MDCK
cell cultures.
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Fig. 3.
Effects of protease inhibitors on IGFBP-5 and IGFBP-6
protease activity in MDCK medium. Aliquots of 48-h-conditioned MDCK
cell medium (25 µl) were incubated with 125I-IGFBP-5 or
-6 (5,000-9,000 counts/min) in the absence of inhibitors ( ) for
10 h at 37°C. 1,10-Phenanthroline (Phe 10 mM), hydroxamic
acid-based metalloprotease inhibitor (TAPI, 0.1 mM), 3,4 dichloroisocoumarin (DCI; 0.2 mM), or aprotinin (Apr; 0.3 µM) was
added as indicated. Reaction products were separated by SDS-PAGE and
visualized by autoradiography. As a control (Co),
125I-IGFBP-5 or -6 was incubated in nonconditioned medium
under identical conditions. The autoradiogram of 1 representative
experiment of 4-6, testing various protease inhibitors, is shown.
The percentage of intact IGFBP-5 or -6 after the incubation was
determined by densitometry and is listed below each lane.
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For further characterization, the IGFBP-6 protease was partially
purified. A fraction containing protease activity with
125I-IGFBP-6 as substrate was precipitated from conditioned
MDCK medium with ammonium sulfate and was bound to DEAE-Sephacel.
IGFBP-6 protease activity was detected in fractions eluted with 0.5 M NaCl (fractions 7 and 8) and with 1.0 M NaCl
(fractions 13-15) (Fig.
4A). The active fractions were
pooled (pools I and III, respectively), as well
as the inactive fractions 9-11 (pool II), and were applied separately to a hydroxyapatite column. No proteolytic activity was found in the flow through, and only a weak
125I-IGFBP-6 protease activity was recovered in fractions
of pool I eluted at 150 mM KPi (I-1). The loss
of IGFBP-6 protease activity might be due to the dialysis of the
fractions eluted or the separation of a cofactor required for
proteolysis. Strong IGFBP-6 proteolytic activity was found in fractions
of pool III also eluted at 150 mM KPi (III-1),
but not in fractions eluted at 300 mM KPi (III-2) (Fig.
4B). Because the IGFBP-6 protease activity in conditioned media was inhibited by the disintegrin metalloprotease-specific inhibitor TAPI, the fractions eluted from the hydroxyapatite column were tested by immunoblotting with rb 119 antibodies directed against
the disintegrin domain of a disintegrin and metalloprotease (ADAM) 12. Under denaturating conditions, a single immunoreactive band at 30 kDa
was found exclusively in I-1 and I-2 but not in the pool
III-eluted fractions (Fig. 4C). When the fivefold
concentrated I-1 and I-2 fractions were analyzed by SDS-PAGE and silver
staining, in addition to the major BSA band, several weak bands of 88, 105, 40, and 34 kDa were detected, but no polypeptide of 30 kDa (not shown). In the initial fraction used for purification, a second disintegrin immunoreactive band at 35 kDa was detected. ADAM 12 S is
the only soluble member of this protease family known so far that is
composed of four domains forming an ~68-kDa mature glycosylated
protease after cleavage of the 25-kDa prodomain (25). To
examine whether the 30-kDa disintegrin immunoreactive band is a
fragment of ADAM 12 S, aliquots of the fraction applied on the
DEAE-column (AD) and of the fractions eluted with low salt concentration (I) were immunoblotted with antibodies against the disintegrin domain (rb 119), the cysteine-rich domain (rb 122), and the
prodomain (rb 132) of human ADAM 12 S. The staining pattern of the two
fractions with the three antibodies shown in Fig.
5 is partially consistent with the
expected sizes of full-length ADAM-12 S (~92 kDa) or the mature
protease (~65 kDa). All polypeptide bands of smaller molecular mass
indicate the presence of truncated fragments. Because these fragments
were found in fractions that lost IGFBP-6 protease activity, it is
likely that they present inactive protease polypeptides. Thus the
30-kDa immunoreactive band may consist of the disintegrin domain (~10
kDa), the cysteine-rich domain (~9 kDa), and at least 11 kDa of the
metalloprotease domain. Larger molecular mass complexes of ADAM 12 S
with other proteins, as detected with the rb 122 antibody, have been
described (25). Because the cross-reactivity of the
anti-human ADAM 12 S antibodies with the polypeptides secreted by MDCK
cells is unclear, the significance of the data is speculative.

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Fig. 4.
Partial purification of IGFBP-6 protease.
A: ammonium sulfate precipitates of conditioned medium from
MDCK cells were dissolved, dialyzed, and applied on a DEAE-Sephacel
column. Bound proteins were eluted by a stepwise NaCl gradient
(top). Dialyzed aliquots of every second fraction were
tested for IGFBP-6 proteolytic activity, followed by SDS-PAGE and
autoradiography (bottom). Underlined fractions were pooled
separately (pools I, II, and III). B:
after dialysis, pools I, II, and III were applied
separately to an hydroxyapatite column. Bound proteins were eluted at
150 mM (1) or 300 mM (2) KPi.
Dialyzed aliquots of these fractions were incubated with
125I-IGFBP-6 for 6 h at 37°C and analyzed by
SDS-PAGE and autoradiography. Co, control incubation of
125I-IGFBP-6 with buffer. AD, aliquots of applied sample on
DEAE-Sephacel. C: aliquots of the same fractions shown in
B, eluted from hydroxyapatite column, were solubilized under
nonreducing conditions and analyzed by SDS-PAGE and anti-disintegrin
immunoblotting with the rb 119 antibody. Elution of proteins from the
DEAE- or hydroxyapatite column in a second purification protocol with
linear NaCl or KPi gradients, respectively, did not result
in better separation of IGFBP-6 protease activities.
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Fig. 5.
Immunoblot analysis of MDCK medium fractions with antibodies
against different ADAM 12 S domains. Aliquots of MDCK medium applied on
the DEAE-ion exchange column (AD) and of the fraction eluted from the
column with 0.35 M NaCl (I) were separated by SDS-PAGE under
nonreducing conditions, transferred to nitrocellulose, and analyzed
with antibodies against the disintegrin domain (rb 119), the
cysteine-rich domain (rb 122), and the prodomain (rb 132) of ADAM 12 S. Immunoreactive polypeptides were visualized by peroxidase-conjugated
secondary antibodies and enhanced chemiluminescence.
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When the IGFBP-6 protease activity was measured in the III-1 fraction
in the presence and absence of protease inhibitors, the proteolytic
activity was inhibited by neither Phe nor by TAPI but was almost
completely blocked by the serine protease inhibitors DCI and Apr (Fig.
6), indicating the separation of the
IGFBP-6 metalloprotease from the serine protease. Of interest,
after incubation of 125I-IGFBP-6 with the fraction III-1, a
prominent fragment of 10 kDa was formed compared with the continuing
proteolytic cleavage in the unfractionated medium.

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Fig. 6.
Effects of protease inhibitors on partially purified
IGFBP-6 protease-containing fraction. Aliquots of the partially
purified fraction III-1 eluted from hydroxyapatite column (see Fig. 5)
were incubated with 125I-IGFBP-6 for 8 h at 37°C in
the presence or absence ( ) of Phe (10 mM), TAPI (0.1 mM), DCI (0.2 mM), or Apr (0.3 µM). Reaction products were separated by SDS-PAGE
and visualized by phosphorimaging. Densitometric evaluation revealed
the percentage of remaining intact IGFBP-6 given below each lane. The
experiment was carried out twice with similar results.
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Polarized sorting of IGFBP-6 protease.
To analyze whether newly synthesized IGFBP proteases are selectively
directed to the apical or basolateral membrane in polarized MDCK cells,
apical and basolateral media conditioned for 24 h were collected
from cells grown on filter inserts 4 days after reaching confluence.
Incubation of 125I-IGFBP-4 with apical medium revealed that
64% of the IGFBP-4 remained intact compared with only 28% of the
125I-IGFBP-4 after incubation in the basolateral medium
(Fig. 7). Similarly, the majority of
IGFBP-6 proteolytic activity was found in the same basolateral medium
(Fig. 7), whereas 54% of the IGFBP-6 remained intact in the apical
medium. These data indicate that proteases cleaving both IGFBP-4 and -6 are secreted preferentially to the basolateral side. For comparison,
filter-grown MDCK cells labeled with [35S]methionine for
4.5 h secrete more newly synthesized proteins to the apical than
to the basolateral medium (~1.8-fold) as measured by TCA-insoluble
radioactivity. The composition and the ratio of labeled proteins
secreted into the apical and basolateral media were different and
polypeptide dependent (Fig.
8A). Thus, when
-hexosaminidase activity was determined, a 2.6-fold (range
2.3-3.1, n = 6) higher activity was measured in
the apical than in the basolateral medium (Fig. 8B). In
addition, conditioned media from the apical and basolateral sides were
analyzed by ligand blotting with 125I-labeled IGF-II. In
both media, one band with an estimated molecular mass of 28 kDa and a
prominent band at 25 kDa were detected, showing an approximate 4.2-fold
higher abundance in the apical (range 3.9-4.7; estimated by
densitometry) than in the basolateral medium (Fig. 8C).
These data indicate that the presence of proteases cleaving IGFBP-4 and
-6 as substrate in the basolateral medium is specific and not due to
loading differences. The identity of the 28- and 25-kDa IGFBPs could
not be determined, because none of the tested antibodies raised against
various human or rodent IGFBPs cross-reacted with canine IGFBPs in
immunoblots, and no hybridization of RNA from MDCK cells with any of
the cDNAs probes specific for human IGFBP-1 to -6 was observed (not
shown).

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Fig. 7.
Polarized secretion of IGFBP proteases. Medium was
collected from the apical (a) or basolateral (b) side of filter-grown
MDCK cells conditioned for 24 h. Aliquots of the media (50 µl)
were tested for protease activity against 125I-IGFBP-4 and
125I-IGFBP-6, followed by SDS-PAGE and autoradiography. As
a control (Co), 125I-IGFBP-4 or -6 was incubated in
nonconditioned medium under identical conditions. Autoradiograms of 1 representative experiment of 2 (IGFBP-4) or 4 (IGFBP-6) are shown.
Aliquots contain 12.2 (a) and 4.9 mU/ml (b) -hexosaminidase activity
secreted within 24 h.
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Fig. 8.
Polarized secretion of proteins. A:
filter-grown MDCK cells were labeled with [35S]methionine
([35S]Met) for 4.5 h at 37°C. Twenty percent each
of the apical (a) and basolateral (b) media, corresponding to 18,000 and 10,000 counts/min TCA-insoluble radioactivity, respectively, were
precipitated with acetone, separated by SDS-PAGE (10%), and visualized
by phosphorimaging. This experiment was repeated twice with identical
results. *Distinct proteins secreted into apical medium; , proteins
preferentially secreted to one side. B: -hexosaminidase
activity was measured in duplicates in apical and basolateral media
from 2 cell cultures in parallel. The experiment was repeated 3 times
with MDCK cells after different passages, resulting in variations of
the absolute activity values but with a reproducible ratio of
activities in the apical and basolateral media. Enzyme activity in the
apical medium is given as a percentage (means ± SD) of
activity in the basolateral medium (5-18 mU/ml and 24 h).
C: media from filter-grown MDCK cells were collected after
24 and 48 h from the apical (lanes 1 and 3)
or basolateral side (lanes 2 and 4) and analyzed
by SDS-PAGE (12.5% acrylamide), 125I-IGF-II ligand blot,
and autoradiography. Positions of molecular mass marker proteins are
indicated. A representative blot of 6 independent experiments is
shown.
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DISCUSSION |
The present study shows that MDCK cells secrete neutral proteases
that cleave IGFBPs either to fragments of defined sizes, like IGFBP-2,
-4, and -5, or to small peptides like IGFBP-6, hardly detectable under
the conditions used. Inhibitor studies and data from the partial
purification of the proteases using IGFBP-5 and -6 as substrates
indicate that at least two classes of proteases, metallo- and serine
proteases, are secreted by MDCK cells with different substrate
specificities. The IGFBP proteases in the conditioned medium separated
by ion exchange chromatography into two fractions are inhibited either
by serine protease inhibitors or by metalloprotease inhibitors such as
1,10-phenanthroline and the hydroxamic acid-based inhibitor TAPI. The
latter has been shown to be a potential inhibitor of tumor necrosis
factor-
-converting enzyme (TACE), a member of the disintegrin
metalloproteases (1, 29). The presence of several
immunoreactive polypeptides, including a 30-kDa protein band in
TAPI-sensitive IGFBP-6 protease fractions detected with specific
antibodies directed against the disintegrin and cysteine-rich domain of
ADAM 12 S, suggests the involvement of disintegrin metalloproteases in
IGFBP-6 proteolysis. These metalloproteases have been reported to
contribute to the proteolysis of IGFBP-3, -4, and -5 in human
pregnancy serum and in cleavage of IGFBP-3 by placental trophoblasts
(17, 22). Recently, the direct interaction with IGFBP-3
and the proteolysis of IGFBP-3 by recombinant ADAM 12 S have been shown
(38). Whether disintegrin metalloproteases are involved in
the proteolysis of IGFBPs in MDCK cells remains to be demonstrated and
requires the sequencing of the 30-kDa immunoreactive polypeptide and
the analysis of the ADAM expression pattern. The observed protective
effect of IGF-II on the proteolysis of IGFBP-6 may be due to the
preferential binding of IGF-II to IGFBP-6 compared with IGFBP-4 or -5, resulting in conformational changes that prolong the degradation rate
rather than a direct effect on the protease.
Whereas the identity of proteases cleaving IGFBP-6 is still unknown and
remains to be determined, IGFBP-6 proteolytic activity was also
detected in acidified conditioned media from NIH-3T3 and HaCaT human
keratinocytes (7, 27). However, the pH optimum, the
complete degradation, and the inhibitor profile suggest that different
acid-activated proteases, including the lysosomal protease cathepsin D,
might be involved in IGFBP-6 proteolysis, either directly or indirectly
by activation of IGFBP-6 proteases. Thus, it is rather unlikely that
these acid-activated proteases play a role in regulation of the
extracellular IGFBP-6 level. Data from mouse fibroblasts partially
deficient for several of the lysosomal enzymes indicate that the
acid-activated proteases may play a role in degradation of endocytosed
IGFBP-3 (3).
The data presented here demonstrate that at least the proteases
cleaving IGFBP-4 and -6 are secreted preferentially to the basolateral
medium of polarized MDCK cells. In contrast, the activity of the
lysosomal
-hexosaminidase and the abundance of IGFBPs in the apical
medium are ~2.6 and 4 times higher, respectively, than in the
basolateral medium. It has been reported that, in the human colon
adenocarcinoma cell line Caco-2, a majority of mannose
6-phosphate-containing lysosomal enzymes accumulate in the apical
medium (41). This polarized distribution results from
selective receptor-mediated uptake of lysosomal enzymes from the
basolateral surface, followed by transcytotic delivery to the
apical side (41). It is still unclear whether the low
IGFBP level in basolateral medium results from increased
proteolysis. The addition of serine protease inhibitors or TAPI
increases, if anything, the abundance of IGFBPs in the apical rather
than in the basolateral medium (unpublished results); however, the stability of the TAPI in hydrous solution at 37°C is low, and long-term toxic effects on cultured cells cannot be excluded (S. Rose-John, personal communication). Recently, Remacle-Bonnet et al.
(33) reported on preferential sorting of IGFBP-2 and -4 to
the basolateral side, whereas IGFBP-6 is primarily delivered to the
apical surface of polarized enterocyte-like HT29-D4 human colonic
carcinoma cells. However, the IGFBP protease activities are not
determined in the media of HT29-D4 cells, and the molecular mechanism
underlying the distinct pattern in IGFBP abundance in media from
polarized cells is not known. It is believed that the transport of
newly synthesized proteins to the basolateral side represents a default
pathway for exocytosis, whereas the sorting to the apical side is
signal dependent, occurring in the trans-Golgi network of
MDCK cells (28, 34). Sorting signals for directing proteins in polarized cells to the apical or basolateral surface are
best characterized for membrane proteins. For MDCK cells, it has been
well documented that glycosylphosphatidylinositol-anchored proteins are specifically targeted to the apical plasma membrane (24). On the other hand, tyrosine and
di-leucine-containing signals have been identified in the cytoplasmic
domains of membrane proteins, which mediate sorting to the basolateral
cell surface (19) and might be followed by a
subsequent transcytotic delivery to the apical membrane.
Furthermore, both N-linked and O-linked glycosylations of proteins, as well as linear glycosaminoglycan chains,
appear to contain apical sorting information (21, 37, 43)
recognized by molecules with lectin activity (13).
However, there is evidence that the sorting mechanism for individual
signals may vary considerably among different epithelial cell types
(19). This might explain the discrepancies between IGFBP
expression in the media of polarized MDCK cells reported in this study
and expression in intestinal HT29-D4 cell media (33).
We propose that a majority of proteases cleaving IGFBP-4 and -6 in epithelial MDCK cells are delivered to the basolateral side and
might be involved in release of IGFs by proteolysis of exogenous
IGFBP · IGF complexes derived from the cellular environment, e.g., mesenchymal cells. Basolateral IGFBP proteolysis is a
prerequisite to guarantee the access of IGFs to cell surface IGF
receptors. A small percentage of IGFBP proteases might be delivered to
the apical side via an indirect pathway, which might control the
steady-state polarized distribution of IGFBPs. In studies with
epithelial MDBK and Caco-2 cell lines, differences in the synthesis and
secretion of distinct IGFBPs and in their mode of regulation have been
reported, which might be necessary for the maintenance of the
proliferative state and/or the initiation differentiation of these
cells (11, 46). Further studies are required to identify
the molecular mechanism of selective IGFBP sorting and to evaluate the
physiological significance in polarized MDCK cells.
 |
ACKNOWLEDGEMENTS |
This work was supported by Deutsche Forschungsgemeinschaft SFB 402 A6 (B. Kübler and T. Braulke), SFB 402 A5 (J.-G. Scharf), and
Graduiertenkolleg 336 (L. Shalamanova).
 |
FOOTNOTES |
Address for reprint requests and other correspondence: T. Braulke, Univ. of Hamburg, Children's Hospital
Biochemistry,
Martinistr. 52, D-20246 Hamburg, Germany (E-mail:
braulke{at}uke.uni-hamburg.de).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 15 November 2000; accepted in final form 16 July 2001.
 |
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