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J Biol Chem, Vol. 275, Issue 8, 5860-5866, February 25, 2000
The Insulin-like Growth Factors (IGFs) I and II Bind to Articular
Cartilage via the IGF-binding Proteins*
Nirav R.
Bhakta ,
A. Minerva
Garcia,
Eliot H.
Frank,
Alan J.
Grodzinsky, and
Teresa I.
Morales§¶
From the Center for Biomedical Engineering and
Department of Electrical Engineering and Computer Science,
Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 and the § Department of Orthopaedic Surgery, Massachusetts
General Hospital and Harvard Medical School,
Boston, Massachusetts 02114
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ABSTRACT |
Bovine articular cartilage discs (3 mm
diameter × 400 µm thick) were equilibrated in buffer containing
125I-insulin-like growth factor (IGF)-I
(4 °C) ± unlabeled IGF-I or IGF-II. Competition for binding to
cartilage discs by each unlabeled IGF was
concentration-dependent, with ED50 values for inhibition of 125I-IGF-I binding of 11 and 10 nM for IGF-I and -II, respectively, and saturation by 50 nM. By contrast, an analog of IGF-I with very low affinity
for the insulin-like growth factor-binding proteins (IGF-BPs),
des-(1-3)-IGF-I, was not competitive with 125I-IGF-I for
cartilage binding even at 100-400 nM. Binding of the 125I-labeled IGF-II isoform to cartilage was competed for
by unlabeled IGF-I or -II, with ED50s of 160 and 8 nM, respectively. This probably reflected the differential
affinities of the endogenous IGF-BPs (IGF-BP-6 and -2) for
IGF-II/IGF-I. Transport of 125I-IGF-I was also measured in
an apparatus that allows diffusion only across the discs (400 µm), by
addition to one side and continuous monitoring of efflux on the other
side. The time lag for transport of 125I-IGF was 266 min,
an order of magnitude longer than the theoretical prediction for free
diffusion in the matrix. 125I-IGF-I transport then reached
a steady state rate (% efflux of total added 125I-IGF/unit
time), which was subsequently accelerated ~2-fold by addition of an
excess of unlabeled IGF-I. Taken together, these results indicate that
IGF binding to cartilage, mostly through the IGF-BPs, regulates the
transport of IGFs in articular cartilage, probably contributing to the
control of their paracrine activities.
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INTRODUCTION |
Insulin-like growth factors
(IGFs)1 are key regulators of
matrix homeostasis in articular cartilage (1, 2). During osteoarthritis (OA), the metabolic balance is lost in favor of degradation, and it has
been suggested that this may be due to an insensitivity of resident
chondrocytes to IGF-I stimulation (3). Recent studies have sought to
understand the underlying mechanisms, and emphasis has been placed on
understanding the role of the IGF-binding proteins (IGF-BPs). This
group of proteins has the ability to modulate the actions of the IGFs,
either enhancing or inhibiting them, depending largely on their
post-translational modifications and tissue localization (for reviews,
please refer to Refs. 4 and 5). An increase in IGF-BP mRNAs in
osteoarthritic compared with normal chondrocytes has been observed (3,
6), and this increased expression is accompanied by increased IGF-BP
proteins in the culture medium of OA cartilage slices (6) or in
monolayer cultures (3, 7). There are also reports of increased IGF-I (8, 9) and IGF type I receptor (7) in OA compared with normal articular
cartilage. These findings are reconciled by the proposal that the
IGF-BPs play an inhibitory role during the disease, blocking the
actions of excess IGF-I (3, 6, 7). While the function of the IGF-BPs in
osteoarthritic cartilage remains to be proven, the role of IGF-BPs in
normal articular cartilage was recently studied in bovine cartilage
organ cultures. These experiments compared the action of native IGF-I
to that of site-mutated analogs of IGF-I with very low binding affinity
for IGF-BPs, but with nearly normal binding to the signaling receptors
(10). The IGF-I analogs were more effective than their native
counterpart in stimulating proteoglycan synthesis when added to
cultured cartilage slices, strongly suggesting that the dominant
endogenous IGF-BP activity was inhibitory to IGF action.
Synovial fluid has a significant concentration of IGFs, ~20-50 ng/ml
in adult normal human (9, 11), and may provide a significant source of
IGFs to cartilage. Chondrocytes express IGF-I mRNA and may also
contribute to the endogenous pool of this growth factor (6, 8). In
adult articular cartilage, single chondrocytes are surrounded by vast
areas of matrix, through which IGFs must be transported to reach a
responsive cell. A general proposal in the literature is that transport
of IGFs from the circulation to extravascular spaces may be controlled
by the IGF-BPs; this may be a mechanism governing paracrine activities
of IGFs within tissues as well. Studies that directly examine binding of IGFs to intact tissue and/or their transport have been very sparse
and have not been fully comprehensive. To enhance our understanding of
the interactions of the IGFs with endogenous tissue sites and the
diffusion of this growth factor through cartilage, we now quantify the
binding of IGFs to bovine articular cartilage, and examine the
involvement of IGF-BPs in this process. We provide a line of evidence
that IGFs bind to articular cartilage discs through IGF-BPs. 1) Binding
of 125I-IGF-I or II to cartilage is specific; 2) an analog
of IGF-I (des-(1-3)-IGF-I) with very low affinity for the IGF-BPs but
normal affinity for the signaling receptor is unable to compete with radiolabeled IGF-I for binding to the cartilage discs, while the native
IGF is a highly effective competitor; and 3) the characteristics of
125I-IGF-I and II binding to the tissue are as expected for
their binding in solution to IGF-BP-6, a major bovine cartilage IGF-BP. In addition, we use a transport apparatus to show directly that IGF
binding contributes to the regulation of radiolabeled IGF transport
through cartilage discs. Our studies provide strong support for the
concept that IGF-BPs are important regulators of the bioavailability of
IGFs in articular cartilage.
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EXPERIMENTAL PROCEDURES |
Materials
Human recombinant (hr) 3-[125I]iodotyrosyl-IGF-I
and II were obtained from Amersham Pharmacia Biotech. Bovine serum
albumin (BSA) was radioimmunoassay grade, from Sigma. hrIGF-I and II
were obtained from R&D Systems, Inc., or from Gropep Pty Ltd (Adelaide,
Australia); hrIGF-I from the latter source was used for studies as
indicated in the text. Des-(1-3)-IGF-I was also from Gropep. The
hrIGF-BP-2 and hrIGF-BP-4 standards were from Austral Biologicals.
Buffers
Phosphate Buffer--
Phosphate buffer consisted of 0.03 M NaH2PO4, pH 4.4, containing
0.02% sodium azide and 0.1% BSA.
Binding Buffer--
Binding buffer consisted of
phosphate-buffered saline (PBS), pH 7.4, containing 0.1% BSA and
protease inhibitors (5.6 µM E-64, 1 mM
Pefabloc SC, 0.7 µg/ml pepstatin A, and 1 mM
orthophenanthroline (o-Phe).
Urea Buffer--
Urea buffer consisted of 0.05 M
Tris maleate, pH 6.0, containing 8 M urea, 0.3 M NaCl, 0.5% Chaps, 5 mM phenylmethylsulfonyl fluoride, 3 mM o-Phe, and 4.5 µg/ml each of
pepstatin A and leupeptin.
Acid Dialysis Solution--
Acid dialysis solution consisted of
4 mM HCl, containing 1 mM o-Phe, 0.1 mM phenylmethylsulfonyl fluoride, and 0.1 µg/ml each of
pepstatin A and leupeptin.
General Procedures
Chromatography and Concentration of Radiolabeled
IGFs--
Immediately prior to use, the lyophilized
125I-IGF was dissolved in 300 µl of 0.01 M
Hac + 0.1% BSA, and run on a 0.6 × 30-cm Sephadex G50 column
equilibrated in phosphate buffer at 14 °C to ensure removal of any
small molecular weight radiolabel. The fractions corresponding to
authentic unlabeled hrIGF standard were pooled, concentrated, and
exchanged into binding buffer in a Centricon-3 filter (Amicon).
Extraction and Isolation of IGF-BPs from Bovine Articular
Cartilages--
This was done essentially as described before (10).
Briefly, slices of bovine articular cartilage from the femoropatellar grooves of adult animals (of approximately 18-24 months of age), or
from the metacarpophalangeal joints of 2-6-week-old calves were diced
into small pieces and extracted in urea buffer at 4 °C. The extracts
were run on DEAE-Sepharose equilibrated in the same buffer to remove
proteoglycans (which bind to the column), the effluent fractions were
pooled, dialyzed against acidic solution, and concentrated in a speed
vacuum centrifuge. Glycosaminoglycan content in the samples was
determined by the dimethylmethylene blue dye binding procedure (12),
and protein was analyzed by the bicinchoninic procedure (MicroBCA), as
directed by the manufacturer (Pierce).
Western Ligand Blotting for IGF-BPs--
The dried samples were
resuspended directly in non-reducing SDS sample buffer at 1 µg of
protein/µl, and 25 µg applied per electrophoresis well. Western
ligand blots were carried out as described previously using
125I-IGF-II as the binding ligand (10, 13).
Cartilage Disc Preparation
Cylindrical plugs of cartilage with the underlying bone slightly
greater than 9 mm in diameter were cored from the femoropatellar grooves of ~18-24-month-old steers. Cartilage-bone plugs were clamped in a sledge microtome and the top ~150 µm of superficial cartilage was removed. Plane parallel discs of 400 µm thickness were
then obtained and subsequently punched to 3 or 9 mm diameter. During
the harvest, tissue was maintained hydrated with PBS supplemented with
2 mM EDTA, penicillin (100 units/ml), streptomycin (0.1 mg/ml), and amphotericin B (0.25 µg/ml).
Equilibrium Binding of IGFs to Bovine Articular Cartilage
Adult bovine articular cartilage (3 mm diameter × 400 µm
thick) was prepared from femoropatellar groove cartilage as described above. After a 10-20-h equilibration in binding buffer, the discs were
transferred to 24-well culture plates containing fresh buffer. In an
effort to randomize any differences between different parts of the
joint, four to five discs were carefully selected, one each from
different anatomical locations, and pooled in each well. 125I-IGF-I or II, as appropriate, was then added to each
well (specific activity 2000 Ci/mmol, an average of ~ 2 × 105 cpm/well, or 0.033 nM
125I-IGF). Graded levels of unlabeled hrIGF-I, hrIGF-II, or
des-(1-3)-IGF-I were then added (or no additions made) as indicated
for each experiment. The incubation volume of 1.5 ml/well was at least
100 times the cartilage volume. Following a 48-h incubation at 4 °C,
each disc was transferred into a wash well containing fresh, IGF-free
binding buffer, removed within seconds, and transferred into a counting vial. The equilibration fluid for each well was counted separately. Immediately prior to weighing, each disc was blotted dry on gauze pads.
In selected experiments, the equilibration fluid was analyzed by
Sephadex G-50 chromatography to determine whether there had been
significant degradation of the 125I-IGF during the
incubation. This was not the case, as >95% of the radiolabel
comigrated with the authentic unlabeled IGF standard (results not shown).
Diffusive Transport Experiments
Cartilage discs (9 mm diameter × 400 µm depth) were
maintained at 4 °C in PBS with inhibitors (as described above) until
mounted in the transport chamber. The transport measurement system has been previously described in detail (14). Briefly, a two-compartment acrylic diffusion chamber (Fig. 5, inset) had ports to fit
up to five cartilage discs (exposed tissue area for transport = 0.38 cm2/disc). Gaskets and O-rings were used to seal the
tissue samples in place so that, when the two chamber halves were
assembled, transport between compartments could occur only through the
tissue. The baths on either side of the cartilage consisted of PBS
supplemented with proteinase inhibitors (2 mM EDTA, 5 mM benzamidine HCl, and 1 mM
phenylmethylsulfonyl fluoride). Temperature was maintained at 20 °C.
Both compartments were open to ambient pressure, and portions of the
downstream bath were recirculated at 1-3 ml/min through a modified
flow-through 125I-radioactivity detector (14). Downstream
radioactivity as measured by the detector was continuously recorded at
6-s intervals by the computer.
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RESULTS |
Identification of Major IGF-BPs in Bovine Articular Cartilage from
the Femoropatellar Groove of Adult Animals--
Previous work (10)
identified IGF-BPs in cartilage obtained from the MCP joints of young
and adult bovines. The MCP cartilage is relatively thin and rests on
curved bone surfaces. To facilitate the preparation of cartilage discs
of 400 µm depth, we used femoropatellar groove (FPG) cartilage from
adult animals, which is relatively thick and rests on a fairly smooth
planar surface. Biochemical variations in cartilages from different
joints, including differential susceptibility to metabolic effectors
have been reported (15), so it was important to determine if the bovine
FPG cartilage had a similar IGF-BP profile as the previously
characterized tissue dissected from MCP joints. To provide a direct
side by side comparison, cartilage slices from each of the two joint
sources (FPG and MCP) were each extracted, purified, and analyzed under
parallel conditions (see "Experimental Procedures"). Fig.
1 shows the Western ligand blot.
125I-IGF-II was used as the binding ligand since it detects
all the IGF-BPs at least as well as the IGF-I ligand (13), and ensures detection of IGF-BP-6, which reacts weakly if at all with
125I-IGF-I under these conditions (10, 13). Lane
1 shows the migration of IGF-BPs from the MCP cartilage, and
lanes 2 and 3 show the binding proteins from FPG
cartilage derived from the joints of two different bovines. As can be
seen, the major IGF-BP in all three lanes migrated to a position above
that of the Mr 21,500 standard, with an apparent
Mr of ~23,000. This protein was previously
identified in MCP cartilage as IGF-BP-6 by Western immunoblotting and
by its marked preferential affinity for IGF-II over IGF-I (10). The
identity of the bands was further verified by lowering the exposure
time of the films to a third of the time shown in Fig. 1 (lower
exposure = 24 h); the typical cartilage IGF-BP-6 doublet
observed in previous studies (10) was evident in the cartilage samples.
The smaller Mr band had a similar migration velocity to the Mr 21,500 standard and a lower
intensity than the larger band in the pair (data not shown). All of the
above data support the identification of this major protein as
IGF-BP-6. An additional band of Mr ~32,000 was
seen in the blots, its migration coinciding with that of the hrIGF-BP-2
standard (lane 5), while being slightly slower than the MCP
cartilage IGF-BP previously identified as IGF-BP-2. Additionally, a
doublet of larger size (Mr ~ 43,500-41,500)
typical of glycosylated IGF-BP-3 was also faintly seen in one of the
samples (lane 3). This IGF-BP has not been definitively
identified in any bovine cartilage, but a doublet of similar size has
been identified in human OA cartilage (3, 6), and more variably in
apparently normal cartilage (3). We concluded from these experiments
that the FPG cartilage contains an IGF-BP pattern very similar to that
identified in bovine MCP cartilage, IGF-BP-6 providing a dominant
signal as assessed by Western ligand blot analysis.
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Fig. 1.
Western ligand blot analysis of extracted
proteins from bovine articular cartilage. Articular cartilage was
dissected from the femoropatellar groove of two adult bovines (joints 1 and 2) and from the metacarpophalangeal joints of a newborn calf
(MCP). The extracts from joints 1 and 2 were prepared using
tissue obtained from the same bovines used for coring discs for
experiments shown in Figs. 3 and 4. Proteins were extracted and
separated from proteoglycans as indicated under "Experimental
Procedures." Twenty-five µg of cartilage protein (or 20 ng of
hrIGF-BP-2 or IGF-BP-4 standards each) were applied on each lane prior
to SDS-PAGE, which was carried out using 14% polyacrylamide gels under
non-reducing conditions. Following transfer to nitrocellulose
membranes, the proteins were allowed to react with
125I-IGF-II, and IGF-binding proteins visualized following
radiography and film exposure for 72 h.
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IGF-I and IGF-II Competition with 125I-IGF-I for
Binding to Cartilage Discs--
Experiments were carried out to test
the effect of graded levels of unlabeled IGF-I as competitor to
125I-IGF-I for binding to cartilage discs (incubation was
for 48 h at 4 °C in the absence of competitor, or in the
presence of 0.1-200 nM IGF-I). Fig.
2A shows the results.
Competition was concentration-dependent with unlabeled IGF-I
concentration and reached saturation by ~50 nM (binding
of 125I-IGF-I remained constant at 12-13% of the total in
the presence of 50, 100, and 200 nM IGF-I, denoting the
unspecific binding component). The ED50 (the point at which
50% of the specific binding of the tracer is inhibited by unlabeled
IGF) was ~11 nM (see inset in Fig. 4 for
a linear plot of the data and dashed lines used to calculate specific binding). Fig. 2B shows the parallel experiment in which the IGF-II isoform was used as the unlabeled binding competitor. Again, there was a concentration-dependent
competition with 125I-IGF-I for binding to the cartilage
discs, with saturation of the response by 50 nM (binding of
the tracer was constant at 11-15% between 50 and 200 nM).
The ED50 was ~10 nM, indicating that IGF-I and IGF-II are equipotent in the competition with IGF-I tracer for
binding to cartilage sites.
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Fig. 2.
Competition of unlabeled IGFs with
125I-IGF-I for binding to bovine articular cartilage.
Articular cartilage discs from the same bovine joint were used in
parallel for the IGF-I (panel A) and IGF-II
(panel B) competition experiments. Each
bar represents the mean of values obtained for five
cartilage discs, and the line on top represents the standard
deviation.
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Competition of Des-(1-3)-IGF-I with 125I-IGF-I for
Binding to Cartilage Discs--
Des-(1-3)-IGF-I is a truncated,
naturally occurring form of IGF-I that has been shown to have an
average of only ~1% of the affinity of native IGF-I for the IGF-BPs,
while retaining affinity of intact IGF-I for the signaling IGF-type I
receptor (16-19). The des-(1-3)-IGF-I variant lacks the 3 amino-terminal amino acids, and systematic studies have shown that
removal of Glu3 accounts for the dramatic loss of binding
affinity for the IGF-BPs. We postulated that if binding of IGF-I to
cartilage occurs through IGF-BP sites, des-(1-3)-IGF-I would be unable
to effectively compete with 125I-IGF-I for binding to this
tissue. In a competition experiment, native hrIGF-I from R & D systems
or from Gropep showed 80-82% inhibition of 125I-IGF-I
binding to cartilage at 50-100 nM (Fig.
3A). On the other hand,
hr-des-(1-3)-IGF-I was unable to compete with the radiolabeled ligand
at any of the concentrations tested, which included the 50-400
nM range (Fig. 3B). This experiment strongly
suggests that the IGF-BPs are involved in the specific binding of IGF-I
to cartilage.
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Fig. 3.
Comparison of the competition of
des-(1-3)-IGF-I and of IGF-I with 125I-IGF-I for binding
to bovine articular cartilage. Panel A shows
the competition of selected concentrations of hrIGF-I with the
radiolabeled IGF-I for binding to cartilage. The hrIGF-I were obtained
from R&D systems or from Gropep, as indicated in the figure.
Panel B shows the lack of competition for
inhibition of 125I-IGF-I binding by increasing
concentrations of the des-(1-3)-IGF-I analog from Gropep.
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Competition of IGF-I and IGF-II with 125I-IGF-II for
Binding to Cartilage--
To further test the hypothesis that IGF-BPs
constitute the major binding sites for IGFs in cartilage, we set up an
additional competition experiment between IGF-II tracer and the two
unlabeled IGFs. The experiment was based on the following
considerations. It is well documented in the literature that IGF-BP-6
is distinctive in that it displays a much higher affinity for IGF-II
than IGF-I in competition experiments carried out in solution when
IGF-II is used as the tracer, but this differential affinity is not as marked when radiolabeled IGF-I is used. The measured potencies of
IGF-II/IGF-I for competition with 125I-IGF-II range between
20 and 70, while the differences are only ~2-fold when
125I-IGF-I is used as the tracer (Ref. 19; for review, see
Ref. 20). Another potentially important cartilage IGF-BP, IGF-BP-2, also displays higher affinity for IGF-II than IGF-I in solution studies, even though the differences on the average are not as pronounced as for IGF-BP-6: the reported potency ratios of IGF-II/IGF-I for IGF-BP-2 competition with 125I-IGF-II vary between 2- and 40-fold (19, 20). We reasoned that because of its endogenous IGF-BP
content (IGF-BP-2 and -6), articular cartilage would probably show a
preferential affinity for IGF-II versus IGF-I when
125I-IGF-II is used as a tracer. When IGF-II was used to
compete with this tracer for binding to cartilage, the ED50
for inhibition was 8 nM (Fig.
4), compared with an ED50 of
160 nM when IGF-I was used as the binding competitor
(potency ratio for IGF-II/IGF-I: 20). Together with the results
obtained with the des-(1-3)-IGF-I analog, these results strongly
suggest that endogenous IGF-BPs in native cartilage constitute a major
pool of binding sites for IGFs in articular cartilage.
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Fig. 4.
Competition of unlabeled IGF-I and IGF-II
with 125I-IGF-II for binding to bovine articular
cartilage. This experiment was similar to that shown in a
bar format in Fig. 2, except that IGF-II is used as the
binding tracer. For comparison, the data of Fig. 2 are replotted in a
linear form as an inset labeled
125I-IGF-I. As indicated in the figure, for both
the inset and the main figure, the  symbols connected by solid lines represent the
competition by IGF-I, and the  symbols connected by solid
lines represent the competition by IGF-II. The upper
horizontal dashed line across the graph indicates the binding of
tracer in the absence of competitor, and the lower
horizontal dashed line denotes the
level at which saturation is reached by the IGF-II competitor (the
unspecific binding component); these lines were used as boundaries to
denote the specific binding component and evaluate the ED50
values for competition by the unlabeled IGFs.
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Transport of 125I-IGF-I through Cartilage
Discs--
These experiments were designed to test the ability of
IGF-I to diffuse through the full depth of the cartilage discs, and to
assess the role of specific binding of the growth factor in this
process. In these series of experiments, consistent with the results of
Fig. 2A, a bolus of 100 ng of IGF-I effectively competed
with radiolabeled IGF-I for binding to cartilage discs in equilibrium
binding experiments. This concentration of IGF-I was used to compete
for binding of 125I-IGF-I in transport experiments. Fig.
5 (inset) shows a diagram of
the transport apparatus. The 125I-IGF-I was added at the
start of the experiment to the "upstream" compartment, allowed to
diffuse through the 400-µm-thick cartilage discs, and the effluent
radioactivity monitored in the "downstream" compartment. A total of
nine transport experiments were carried out with very reproducible
trends. Fig. 5 shows a representative experiment in which the efflux of
125I-IGF-I into the downstream bath (as a percentage to the
total added to the upstream compartment) is plotted as a function of time. The time lag ( lag) before effluent counts were
observed after addition of 125I-IGF-I to the upstream
compartment was calculated to be 266 min, using the intercept on the
time axis for the line fit to the first steady state (linear) portion
of the data (t = 400-620 min). In general, the time
lag reflects the combined diffusion-reaction transport process
governing emergence of a solute into the downstream bath (21). In this
case, the lag would include the time that it takes for endogenous IGF
sites to be saturated with the radiolabeled growth factor, after which
a steady state rate of 125I-IGF-I diffusion is seen between
400 and 620 min. The diffusivity for IGF-I calculated from this steady
state flux was 4.1 × 10 7 cm2/s
(calculation of this diffusivity took into account the small amount of
residual low molecular weight iodinated species in the 125I-IGF-I preparation, by means of separate control
experiments as described in detail by Garcia et al. (14)).
The diffusivity was used to estimate the lag that would
be expected in the absence of binding (21). This theoretical
lag is 11 min, 24-fold faster than experimentally
observed, strongly suggesting that transport of IGF-I through these
cartilage discs was dramatically slowed by binding of the growth factor
to sites within the tissue. To further test this possibility, a bolus
of unlabeled IGF-I was then introduced into both the upstream and
downstream compartments after the steady state rate of efflux had been
achieved, at t = 620 min. The concentration of
unlabeled IGF-I, 1 × 107 M (100 nM), was at least 100-fold higher than that of labeled material initially present in the upstream compartment. This resulted in a nearly 2-fold rate of increase in the efflux of
125I-IGF-I at t = 620 min (extrapolated from the
initially linear portion of the curve), followed by a return to the
previous steady state flux by 1400 min. This latter observation
reflects the exchange of bound 125I-IGF-I for unlabeled
IGF-I during the 195 min of accelerated linear efflux of radiolabeled
material into the downstream compartment, followed by progressive
saturation of these sites by unlabeled IGF-I. Addition of a second
bolus of IGF-I at 1450 min did not further affect the rate of efflux,
suggesting previous saturation of binding sites by the unlabeled growth
factor added at t = 620 min. The binding properties
exhibited by the articular cartilage discs during the non-equilibrium
transport of IGF-I, particularly the competition for tissue sites by
unlabeled IGF-I, are consistent with the role of the IGF-binding
proteins delineated in the equilibrium binding studies.
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Fig. 5.
Diffusion-reaction kinetics and steady state
diffusion flux of 125I-IGF-I across cartilage.
Inset, transport chamber. The cartilage discs are sealed
using gaskets and O-rings to assure that all solute and fluid transport
occurs across the tissue between the two compartments. Fluid in both
compartments is magnetically stirred and recirculated with return ports
close to the tissue in order to further minimize stagnant layer effects
on transport (for further details, please refer to "Experimental
Procedures" and Ref. 14). Main panel, normalized
downstream concentration of 125I-IGF-I is plotted as a
function of time. A bolus of radiolabeled IGF-I was introduced
into the upstream compartment at time, t = 0 (indicated
by the left-hand side arrow). The time at which
measurable amounts of 125I-IGF-I were seen in the
downstream compartment is given by Tlag = 266 min. At t = 620 min, unlabeled IGF-I was added to bring
both compartments up to 107 M
(middle arrow), resulting in an exchange of the
unlabeled IGF-I with radiolabeled IGF-I reversibly bound to the tissue,
followed by an eventual return to the previous steady state flux
(evident between 1200 and 1400 min). At t = 1450 min,
another bolus of unlabeled IGF-I was added to both compartments
(right-hand arrow), and no change in
flux was detected. Cd, concentration in downstream
bath; Cµ, concentration in upstream bath.
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DISCUSSION |
Previous studies have shown that IGFs bind to various types of
cells and their extracellular matrices produced in culture through
IGF-BPs. In this study, instead of examining IGF binding to tissue
structures produced in vitro, we examined the specific binding of IGFs to articular cartilage discs, and their transport across their depth. These tissues have an architecture largely assembled in vivo. The advantage of this approach is that it
ensures that the tissue characteristics, including its porosity,
three-dimensional molecular structures and interactions, local ionic
character, etc., closely resemble the physiological situation. We
present a line of evidence in support of the specific binding of IGFs to bovine articular cartilage through the endogenous IGF-binding proteins. First, the equilibrium binding experiments clearly showed dose-dependent, saturable competition of excess unlabeled
IGFs with radiolabeled IGF for binding to cartilage discs. Second, the
IGF-I analog, des-(1-3)-IGF-I, which has a very low affinity for
IGF-BPs (an average of ~1% of normal) but normal affinity for the
IGF type I signaling receptor (16-19), is not able to compete with
125I-IGF-I for binding to cartilage, supporting the notion
that the radiolabeled IGF-I binds to cartilage through IGF-BPs. Third, when 125I-IGF-II is used as the binding ligand for
articular cartilage, unlabeled IGF-II displays greater effectiveness as
a competitor for displacement of binding than IGF-I (potency of
IGF-II/IGF-I: 20). This differential affinity is not observed when
125I-IGF-I is used as the binding tracer. These
observations are consistent with the expected solution binding activity
of IGF-BP-2, and particularly IGF-BP-6 to the IGFs (20). Since these
are major IGF-BPs in bovine cartilage (Ref. 10, Fig. 1), the findings provide evidence that binding occurs through tissue IGF-BPs.
It is important to note that, while the local tissue environment could
affect the binding parameters in many ways, the endogenous IGF-BPs
(presumably mostly IGF-BP-6) retain their preferential affinity for
IGF-II; this points to an important role for this isoform in the
tissue. A proposed model is that that IGF-BP-6 has two binding sites
for IGFs. One has weak affinity for the two IGFs, while the other has a
much greater affinity for IGF-II than I (19). In binding studies, at
low levels of IGF-II tracer, this second high affinity site may be
preferentially filled and preclude binding to the other site. The only
other protein in cartilage known to have preferential affinity for
IGF-II over IGF-I is the IGF-II/M-6-P receptor. The affinity of this
receptor for IGF-I is 100-500-fold or less that for IGF-II (4, 22). In
our studies, the affinity of IGF-I was only ~20-fold lower than that
of IGF-II (based on ED50 values for competition of the ligands with 125I-IGF-II), which is in the range of
reported affinities of IGF-BP-6 for IGFII/IGF-I (potency ratio of
20-70) using this tracer (20). A slight contribution of the IGF-II
receptor to the experimental observations cannot be strictly ruled out.
The results presented in this paper differ significantly from a
previous proposal that IGFs bind to cartilage unspecifically through
proteoglycans (23). In agreement with our study, this previous work
showed a linear uptake of radiolabeled IGFs by cartilage, but the
authors did not use a specific competitor, and provided calculations
based on the assumption that only signaling receptors would be
responsible for specific binding. The present study demonstrates that
most of the binding of IGF to the tissue is specific and strongly
suggests the involvement of endogenous IGF-BPs in this process. The
results are not inconsistent with the possibility that the IGF-BPs may
in turn be bound to proteoglycans in cartilage. Heparan sulfate
proteoglycans have been implicated in IGF-BP binding to extracellular
matrices (24-26). IGF-BP-6 contains a peptide sequence (YRKRQCRS)
within its thyroglobulin repeat region that conforms to the consensus
sequence XBBBXXBX (X = non-basic amino acid, B = basic) for heparan sulfate binding in a
number of proteins, and the IGF-BP-6 sequence has been directly shown
to bind to heparin (24). IGF-BP-2 has a motif that also displays high
heparin binding activity (BBXB) in many proteins, and it has
been shown that this IGF-BP binds to heparin in the presence of IGF-I,
suggesting that a cryptic heparin binding site may be exposed following
binding of the IGF-BP to IGF (25). Thus, a candidate anchor for the IGF-BPs is perlecan, a heparan sulfate proteoglycan present in the
pericellular matrix of articular cartilage (27). It is also of note
that IGF-BP-2 has been found to bind aggrecans through the chondroitin
sulfate chains (28), but the specificity of this interaction remains to
be defined. The localization of the IGF-BPs in articular cartilage will
be the subject of a future study.
It is worth pointing out that the present binding studies were carried
out under free-swelling, non-loading conditions. It is possible that
either dynamic or static compression may alter the affinities of the
tissue for the IGFs by altering the tissue structures to which they are
bound, by water extrusion, fluid flow, fluid-generated electrical
currents, matrix compaction, etc. (29, 30). Thus, under selected
conditions, for example, under dynamic compression, the IGF-BPs may
provide a large reservoir of IGFs that is displaced from its binding
sites and made accessible to signaling receptor on the cell surface. In
other words, the IGF-BPs may be inhibitory under unloaded conditions,
but may aid IGF action under loading conditions. Future studies will
address these issues.
In this study, we also present evidence for the involvement of the
IGF-BPs in the regulation of diffusive transport of IGF-I through
cartilage. There are only a few reported studies on the transport of
the IGFs in the general literature, and these have focused mostly on
vascular to intravascular transport. Both in vivo and
in vitro models have been used to analyze IGF transport, and
variable results have been obtained with regards to the role of IGF-BPs
in this process. For example, a study of IGF transport from the
vasculature to intestinal tissue concluded that IGF-BPs were not
primarily involved in this process (31), and a study of radiolabeled
IGF-I diffusion through endothelial cell monolayers determined that
this process was paracellular, and not affected by competition with
unlabeled IGFs (32). On the other hand, a recent study of an in
vivo model of skin wound repair using Hunt-Schilling chambers in
rats measured the clearance of exogenous growth factors from the wound
into the circulation, and concluded that IGF-BPs were involved in the
regulation of clearance (33). The rates of clearance for insulin and
for LR3-IGF-I were faster than those for IGF-I or II, and
this was equated with a reduced level of binding to wound fluid IGF-BPs
for the faster ligands. These interesting studies provide impetus for dissecting out various processes underlying the complexities of transport through tissue and body spaces. To our knowledge, the transport kinetics of IGFs through a tissue layer of fairly homogeneous structure, and the role of IGF-BPs in this process, had not been directly examined. The articular cartilage discs prepared for the
present study were of fairly uniform tissue architecture and suitable
for reproducible experimental analysis; they allowed measurement of
binding of the two IGF isoforms and direct analysis of the transport of
IGF-I across their 400-µm depth.
We provide quantitative evidence in support of the regulation of IGF
transport through cartilage by specific binding of the growth factor to
the tissue. First, the time lag for transport of radiolabeled IGF-I
through cartilage discs and the kinetics of the measured diffusive
transport, combined with theoretical calculations (21), pointed to the
existence of binding sites in the tissue that initially slow the
transport of IGF through the tissue. A likely explanation for this
observations is that during the time lag for 125I-IGF-I
efflux into the downstream bath, available sites in the tissue
gradually attain reversible binding equilibrium with the concentration
of radiolabeled ligand, and that the subsequent linear, steady state
rate of efllux (by t ~ 600 min) mostly reflects the
rate of unimpeded 125I-IGF diffusion through tissue spaces.
Second, this steady state rate of radiolabeled IGF transport can by
enhanced nearly 2-fold by addition of unlabeled IGF to both the
upstream and downstream compartments. This likely represents the
release of bound 125I-IGF-I from tissue sites by exchange
with the unlabeled ligand entering the tissue from both sides, combined
with the continued diffusion of radiolabeled IGF through the tissue.
The experimental findings from these transport experiments, together
with the binding competition data of Figs. 2-4 suggest that specific
binding of IGFs to cartilage (mostly through the IGF-BPs) regulate the
paracrine activities of the IGFs in articular cartilage.
| |
ACKNOWLEDGEMENTS |
We gratefully acknowledge the valuable
scientific input of Dr. Douglas A. Lauffenburger (Massachusetts
Institute of Technology), and the consultantship of Dr. Stephen Trippel
(Massachusetts General Hospital and Harvard Medical School) during the
IGF transport experiments.
| |
FOOTNOTES |
*
This work was supported by a supplemental award to National
Institutes of Health Grant R01 AR33236 and by the Center for Biomedical Engineering (Massachusetts Institute of Technology, Cambridge, MA).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.
¶
To whom correspondence should be addressed: Dept. of
Orthopaedic Surgery, White Bldg. R426, Massachusetts General Hospital, Boston, MA 02114. Tel.: 617-724-7397; Fax: 617-724-7396; E-mail: tmorales@partners.org.
| |
ABBREVIATIONS |
The abbreviations used are:
IGF, insulin-like
growth factor;
IGF-BP, IGF-binding protein;
Chaps, 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonic acid;
OA, osteoarthritis;
PBS, phosphate-buffered saline;
BSA, bovine serum
albumin;
o-Phe, orthophenanthroline;
MCP, metacarpophalangeal;
FPG, femoropatellar groove.
| |
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ABSTRACT
INTRODUCTION