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INTRODUCTION |
Recent evidence suggests that the binding of growth factors
to the extracellular matrix
(ECM)1 may be a major
mechanism for regulation of growth factor activity (for review, see
Ref. 1). It has long been thought that growth factors released from
bone matrix play important roles in the coupling of bone resorption to
bone formation and in repair processes such as fracture healing.
However, at present little is known about the mechanisms by which
growth factors are stored in the ECM, the ECM proteins with which they
interact, or the molecular mechanisms by which they are released.
Bone ECM is the major storage site in the body for TGF-
(2, 3). This
ECM-bound TGF-, which is predominantly the TGF-1 isoform, is
stored in a latent form and can be released and activated by resorbing
osteoclasts (4). Once released from the matrix and activated, TGF-
can influence many of the steps in the remodeling pathway, including
inhibition of osteoclast formation and activity, stimulation of
recruitment and proliferation of osteoblast precursors, and stimulation
of mature osteoblasts to produce bone matrix proteins (reviewed in Ref.
5). TGF- has therefore been implicated as a coupling factor that
coordinates the processes of bone resorption and subsequent bone
formation. Although it is known that bone matrix-bound TGF- can be
released by osteoclasts (4) and that osteoclasts are capable of
activating both matrix-derived and exogenously added latent TGF-
(6-8), the molecular mechanism(s) by which TGF- is released from
ECM-bound stores remain unclear.
We have shown previously (9, 10) that a major mechanism for storage of
latent TGF- in bone matrix is via its association with the latent
transforming growth factor--binding protein-1 (LTBP1). LTBP1 is a
member of an emerging superfamily of ECM proteins, which includes
fibrillins 1 and 2 and LTBPs 1-4 (reviewed in Refs. 11 and 12). LTBP1
was originally identified as a component of the large latent TGF-1
complex (13, 14). This complex consists of the 25-kDa mature TGF-
homodimer, which is cleaved from but remains non-covalently associated
with a 75-kDa portion of the propeptide homodimer (also known as
latency-associated peptide or LAP). The propeptide is then
disulfide-linked to the 190-kDa LTBP1 (13, 15). Although most cells
produce latent TGF- in the large latent complex, a small latent
complex that lacks LTBP1 and consists of TGF- non-covalently
associated with its propeptide has also been reported as a naturally
occurring form in bone and kidney (5, 16).
LTBP1 facilitates secretion of latent TGF- (17) and may also
modulate activation of latent TGF- (18). More recently it has been
described as a stable component of the extracellular matrix that is
important in storage of latent TGF- in the matrix and may be a
structural component of connective tissue microfibrils (9, 10, 19, 20).
Several studies have suggested that in addition to its role in
storage of latent TGF- in the ECM, LTBP1 may function as a vehicle
for release of latent TGF- from matrix-bound stores. Thus, purified
proteases, such as plasmin and elastase, have been shown to release
TGF- from the matrix of fibroblasts and osteoblasts via cleavage of
LTBP1 (9, 21). However, it is unclear whether this is a phenomenon
associated with pathologically high concentrations of proteases or
whether proteolytic cleavage of LTBP1 represents a true physiologic
mechanism for release of ECM-bound TGF-.
In the present study, we characterized the forms of latent TGF-
produced by primary bone cells and stored in the ECM. Next, by using
osteoclast cell culture systems we investigated the molecular mechanism(s) by which TGF- is released from bone ECM-bound stores during bone resorption. In particular, we examined whether proteolytic cleavage of LTBP1 may be a potential cellular mechanism for the release
of TGF- bound to bone ECM, and we determined whether proteases known
to be important in osteoclast function may be involved in this process.
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EXPERIMENTAL PROCEDURES |
Reagents--
Plasmin, elastase, aprotinin, leupeptin, and
pepstatin A were purchased from Roche Molecular Biochemicals.
Phenylmethylsulfonyl fluoride and E64 were purchased from Sigma. Matrix
metalloproteinases (active MMP2 and MMP9) and the tissue inhibitor of
the matrix metalloproteinases (TIMP1) were purchased from Oncogene
Research Products (Cambridge, MA). 1,25-Dihydroxyvitamin D3
(1,25-D3) was purchased from Biomol (Plymouth Meeting, PA).
Parathyroid hormone-related peptide (PTHrP) was purchased from Bachem
(Torrance, CA).
Antibodies--
Two different rabbit polyclonal antibodies
against LTBP1 were used: Ab39 that recognizes mouse, rat, and human
LTBP1 (kindly supplied by K. Miyazono, Japanese Foundation for Cancer
Research, Tokyo, Japan), and a second antibody, termed the "LTBP1
hinge antibody," raised against a synthetic peptide corresponding to residues 721-744 of the rat LTBP1 sequence (GenBankTM
accession number M55431) that recognizes mouse and rat LTBP1. The
specificity of these antibodies has been described elsewhere (10, 13,
20). A mouse monoclonal antibody against human LTBP1, as well as
neutralizing antibodies to TGF-1 and -2, a pan-specific
TGF--neutralizing antibody, and a goat polyclonal antibody against
latent TGF-1 propeptide were purchased from R & D systems
(Minneapolis, MN). The detection antibody for Western blotting was a
peroxidase-conjugated donkey anti-rabbit (Amersham Biosciences).
Cell Culture--
Tissue culture reagents were purchased from
Invitrogen or JRH Biosciences (Lenexa, KS). Fetal rat calvarial
osteoblasts (FRC) were isolated as described previously (9, 10). The
cells were stored for up to 1 year using standard cryopreservation
procedures and used for experiments after one further passage. UMR-106
cells were a gift from T. J. Martin (St. Vincent Institute of
Medical Research, Fitzroy, Victoria, Australia). TMLC-C32 cells were a gift from D. B. Rifkin (New York University, New York).
Rabbit marrow cells were isolated using a modification of the method of
Tezuka et al. (22). Femora, humeri, ulnae, and radii of
11-day-old New Zealand White rabbits were removed. Connective tissues
were dissected away, and the bones were minced in 
-modified minimal
essential medium (MEM). The bone pieces were vortexed to dissociate
the cells, and bone particles were removed by sedimentation under
gravity. The supernatant was centrifuged, and the cells were
resuspended in MEM supplemented with 10% fetal bovine serum (FBS),
2 mM L-glutamine (LG), and 100 units/ml
penicillin/streptomycin (P/S). The cells were then seeded into 12-well
plates coated with 35S-labeled bone ECM (see below).
Avian osteoclast precursors were isolated as described elsewhere from
the medullary bone of egg-laying White Leghorn hens fed on a low
calcium diet (23). The cells were plated in 150-mm Petri dishes as
described elsewhere (23), and after overnight incubation, adherent
cells were harvested by treatment with 2 mM EDTA in PBS for
5 min at 37 °C. The cells were then replated into 12-well plates
coated with 35S-labeled bone ECM (see below) in MEM
containing 2.5% FBS, 2.5% chicken serum, 2 mM LG, 100 units/ml P/S, and 6 µg/ml arabinose--D-cytosine furanoside. These cells have been shown to fuse and become
multinucleated in the presence of 1,25-D3 (23) (see also
Fig. 5, e and f). The cells were cultured for up
to 6 days, with or without 1,25-D3 and/or protease
inhibitors as described below. Culture media were changed every 2 days.
Fast Pressure Liquid Chromatography (FPLC) Analysis--
FPLC
fractionation was performed as described previously (24). FRC cells
were grown in 150-cm2 flasks in MEM supplemented with
10% FBS, 2 mM LG, 100 units/ml P/S, and 30 µg/ml
gentamycin. At confluence the media were changed to MEM,
supplemented as above but with the addition of 50 µg/ml ascorbic acid
and 3 mM -glycerophosphate (-GP) and the reduction of
the serum to 5%. Thereafter the media were changed every 3 days.
Conditioned medium was harvested from four time points representing sequential stages in the in vitro differentiation of FRC
cells to form mineralized bone-like nodules. The "pre-confluent"
stage was from proliferating cultures at 90% confluence; the "early post-confluent" stage was from 2-day post-confluent cultures; the
"nodule-forming" stage was from 6-day post-confluent cultures in
which multilayered cellular nodules had formed, but were not yet
mineralized; and the "mineralization" stage was from 14-day post-confluent cultures in which mineralized bone nodules were present
(see Fig. 1, inset micrographs). For collection of
conditioned media, phenol red-free Dulbecco's modified Eagle's medium
was used containing 2 mM LG, 100 units/ml P/S, 0.1% bovine
serum albumin, 50 µg/ml ascorbic acid. 25 ml of conditioned medium
was collected per flask over a 48-h culture period. A total of 150 ml
of conditioned medium per time point was concentrated 10-fold over a
50-kDa cut-off membrane using a minisette concentrator (Millipore,
Bedford, MA), and the samples were lyophilized and reconstituted with
1-2 ml of distilled water, pH 7.2. They were then dialyzed against 20 mM tris buffer, pH 7.2, and applied to an analytical Mono-Q
anion exchange column (Amersham Biosciences). The column was eluted with a linear gradient of 0-0.5 M NaCl/20 mM
tris buffer. Fractions were tested for TGF- activity using the
alkaline phosphatase microassay as described below.
TGF- Measurement--
TGF- was measured as described
previously by using either the ROS 17/2.8 microassay (24), which
measures stimulation of alkaline phosphatase activity by TGF- in ROS
17/2.8 osteosarcoma cells, or by using the mink lung epithelial cell
luciferase bioassay (25), which measures stimulation of activity of the
plasminogen activator inhibitor-1 promoter by TGF-. To determine the
total (active + latent) TGF- levels, the samples were acidified to pH 2 using 1 M HCl and then reneutralized using 1 M NaOH immediately before addition to the assay plate.
Latent TGF- values were determined by subtracting active TGF-
measurements from total TGF-.
Pulse-Chase Metabolic Labeling and Immunoprecipitation--
To
examine further the secreted and matrix-bound forms of latent TGF-
produced by FRC cells, pulse-chase metabolic labeling and
immunoprecipitation were performed. Cells were plated into 12-well
multiwell plates at 10,000 cells/cm2 growth area. At 90%
confluence, the cells were washed twice in PBS and incubated for 1 h in cysteine-free MEM supplemented with 5% dialyzed FBS, 2 mM LG, 100 units/ml P/S, 50 µg/ml ascorbic acid. The
cells were then labeled for 30 min using 100 µCi/well [35S]cysteine (Amersham Biosciences) in cysteine-free
MEM supplemented with 5% dialyzed FBS and additives as above. This
was followed by a "cold chase" in complete MEM (containing 0.1 mg/ml L-cysteine), supplemented with 5% FBS and additives
as above for a time course of 15 and 30 min, and 2, 6, 24, and 48 h.
Conditioned media were harvested and protease inhibitors added (1 mM phenylmethylsulfonyl fluoride, 50 µg/ml aprotinin, 1 µM pepstatin A, 10 µM leupeptin). The cells + matrix were washed twice in ice-cold PBS and then lysed in ice-cold
radioimmunoprecipitation buffer (RIPA) (50 mM Tris, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate). The
deoxycholate-insoluble material (essentially matrix) was washed two
more times in ice-cold PBS, transferred to a 1.5-ml tube, and
centrifuged for 5 min at 14,000 rpm. The pellet was then digested for
2 h at 37 °C on an end-over rotator in 500 µl of a solution
consisting of 0.2 units/ml plasmin in plasmin digestion buffer (see
under "Protease Digestions"). This digestion releases any LTBP1
that may still be bound to the ECM (9, 21).
LTBP1 and TGF- in the supernatant and in the plasmin digest of the
matrix were determined by immunoprecipitation followed by SDS-PAGE and
autoradiography as described previously (9, 24) using a rabbit
polyclonal antibody specific for LTBP1 or using a goat polyclonal
antibody specific for LAP.
Preparation of Bone ECM--
To prepare bone ECM for culturing
with osteoclasts, FRC cells were plated into 12-well plates as
described above. At confluence, the medium was changed to MEM
supplemented with 5% FBS, 2 mM LG, 100 units/ml P/S, 30 µg/ml gentamycin, 50 µg/ml ascorbic acid, and 3 mM
-GP. Thereafter, the culture media were changed every 3 days.
Cultures were maintained for 10-14 days, by which time mineralized
bone nodules could be seen, and a thick ECM layer had formed. For
preparation of ECM-coated plates, a modification of the method of Jones
et al. (26) was used. The cells were washed twice in
ice-cold PBS and lysed in PBS containing 0.5% Triton X-100 for 10 min
at 4 °C. This leaves a membrane-like layer of bone ECM adhered to
the bottom of the culture well. The matrix layer was then washed twice
in ice-cold PBS and treated with ice-cold 50 mM ammonium
acetate, pH 7.5, for 10 min at 4 °C (the pH was reduced from 9.0, as
described in Jones et al. (26), to prevent activation of
TGF- by the alkaline pH). The matrix layers were then washed 4 times
in PBS, air-dried, and stored at 
70 °C.
Measurement of Release of LTBP1 and TGF- from
[35S]Cysteine-labeled Bone ECM--
An
immunoprecipitation-based assay was developed for examining release of
bone matrix-bound LTBP1 and latent TGF- by cells cultured on bone
ECM (27). FRC cells were cultured in 12-well plates as described above
until 10-14 days of culture. The cells were then labeled for 48 h
using 100 µCi/well [35S]cysteine in MEM containing
one-fifth the normal cysteine content and supplemented with 5%
dialyzed FBS, 50 µg/ml ascorbic acid, 3 mM -GP, 100 units/ml P/S, 2 mM LG. The labeled bone ECM was prepared as
described above.
To examine release of LTBP1/TGF- by osteoclasts, two sources of
osteoclast precursors were used, rabbit marrow cells and avian
osteoclasts. The cells were plated onto the radiolabeled ECM at
2.5 × 106 cells/well (rabbit marrow cells) or 5 × 106 cells/well (avian osteoclast precursors) in 2 ml of
MEM supplemented as described above. The cells were incubated with
or without addition of 1,25-D3, PTHrP, or protease
inhibitors as specified under "Results." Release of LTBP1 and
latent TGF- was monitored by immunoprecipitating the labeled protein
fragments released into the culture media using anti-LTBP1 antibodies
(the LTBP1 antibodies will precipitate both free LTBP1 and LTBP1
complexed to latent TGF-). Because the osteoclasts themselves were
never exposed to [35S]cysteine, any radiolabeled proteins
present in the culture supernatants were assumed to have been released
from the pre-labeled matrix. The supernatant was transferred to a fresh
tube and protease inhibitors added as described above. The
deoxycholate-insoluble pellet was digested in 0.2 units/ml plasmin as
described above. LTBP1 in the supernatant and in the plasmin digest of
the matrix was then determined by immunoprecipitation with anti-LTBP1
antibodies, as described above.
Protease Digestions--
Plasmin and elastase digestions of ECM
preparations were performed in a digestion buffer consisting of 0.1%
1-O-n-octyl--glucopyranoside, 3 mM
MgCl2, 3 mM CaCl2, 150 mM NaCl, 10 mM Tris, pH 8. MMP2 and MMP9
digestions were performed in MMP digestion buffer (50 mM Tris, 0.2 M NaCl, 5 mM CaCl2,
0.02% Brij 35, pH 7.6). Activity of the MMPs was confirmed in all
experiments by gelatin zymography.
Western Blotting--
Samples were separated on SDS-PAGE using
4-20% gradient gels, transferred onto nitrocellulose, and
immunoblotted as described previously (9, 10). The immunostained bands
were visualized using the ECL detection system according to
manufacturer's instructions (Amersham Biosciences). The immunostained
Western blots were then exposed to X-Omat AR film (Eastman Kodak).
Immunofluorescent Staining--
For immunofluorescent staining,
FRC cells were cultured on lab-tek 8-chamber slides at 10,000 cells/cm2 growth area and maintained for 10-14 days as
described above. ECM was prepared as described above and incubated with
or without proteases as described above. After incubation in proteases,
the ECM layers were fixed in 95% ethanol and stained by
immunofluorescence as described previously (10).
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RESULTS |
Characterization of Secreted Latent TGF- Complexes Produced by
Primary Bone Cell Cultures--
As matrix laid down by primary
osteoblast cultures was to be used to examine release of bone
matrix-bound TGF- by osteoclasts, it was first necessary to
characterize the latent TGF- complexes produced by the osteoblast
cultures and determine what proportion of the latent TGF- was
complexed to LTBP1. Fig. 1 shows results from Mono-Q FPLC analysis of conditioned media samples from FRC cultures at the pre-confluent, early post-confluent,
nodule-forming, and mineralizing stages (see "Materials and
Methods"). The insets in Fig. 1 show the appearance of the
cultures at each of these stages. Two major peaks of latent TGF-
activity were observed, one eluting at 0.22 M NaCl (peak
II), which is the expected elution position for the 100-kDa small
latent TGF- complex (16, 24), and the other eluting at 0.3 M NaCl (peak III), which corresponds to the 290-kDa large
latent TGF- complex, containing LTBP1 (16, 24). The pre-confluent
and early post-confluent cultures produced predominantly the small
latent TGF- complex (peak II). However, with maturation in culture,
the cells switched to producing larger amounts of the 290-kDa
(LTBP1-containing) complex (peak III), which made up ~40% of the
total latent TGF- secreted. In addition, a minor peak eluting at
0.05 M NaCl (peak I) was also observed in nodule-forming
and mineralizing cultures. At present the nature of this peak is
unknown. Antibody neutralization studies indicated that the latent
TGF- activity was predominantly TGF-1 in all three peaks with
smaller amounts of TGF-2 (data not shown). In the pre-confluent and
early post-confluent cultures, peak III showed ~20-30% TGF-2
activity, in contrast to the nodule-forming and mineralizing cultures,
which showed no detectable TGF-2 activity in this peak (data not
shown).
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Fig. 1.
FPLC profiles of latent
TGF- complexes in conditioned media of FRC
cells at sequential stages of differentiation using a Mono-Q anion
exchange column with a linear 0-0.5 m NaCl gradient.
TGF- activity was measured using the ROS 17/2.8 alkaline phosphatase
microassay. Major peaks of latent TGF- activity were seen at 0.22 M NaCl (peak II) and 0.3 M NaCl
(peak III) and a minor peak was seen at 0.05 M
NaCl (peak I). The insets are phase contrast
photomicrographs indicating the morphology and mineralization of the
cultures at the various stages of differentiation (bar, 50 µM).
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Analysis of Matrix-bound Latent TGF- in Primary Bone Cell
Cultures--
LTBP1 has been shown to be cross-linked in the ECM via
the action of transglutaminase (28) and is highly insoluble. Previous studies have shown that proteases such as plasmin and elastase can
release proteolytic fragments of LTBP1 from the ECM (9, 21) and that
plasmin can activate latent TGF- (29, 30). These serine proteases
were therefore used to examine the ECM-bound forms of latent TGF- in
primary bone cell cultures (see Fig. 2).
Treatment of 35S-labeled bone ECM with plasmin or elastase
resulted in release of radiolabeled products, which could be
immunoprecipitated with antibodies against LTBP1 (see Fig.
2a). Plasmin released a doublet at ~110-130 kDa
(black arrowhead), corresponding to cleaved fragments of
free LTBP1, as well as a minor band at ~230 kDa (white
arrowhead), which is the expected size of the large latent TGF-
complex containing cleaved LTBP1. Elastase released similar fragments
but appeared to cleave LTBP1 at a different site, as evidenced by a
faster migration. As expected, in both cases the upper band,
corresponding to the large latent TGF- complex, disappeared under
reducing conditions (data not shown). To confirm that the upper band at 230-kDa contained TGF-, supernatants from plasmin-digested bone ECM
were immunoprecipitated with antibodies to LTBP1 and to the latent
TGF-1 propeptide (also known as latency-associated peptide or LAP)
to demonstrate co-migration (see Fig. 2b).
Immunoprecipitation with anti-LAP antibodies produced a band at ~230
kDa (white arrowhead), which exactly co-migrated with the
upper band precipitated with anti-LTBP1. An additional band was
observed at ~200 kDa, presumably representing latent TGF- bound to
an as yet unidentified protein.
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Fig. 2.
Release of LTBP1 and large latent
TGF- complex from bone matrix by digestion
with plasmin (0.1 units/ml for 2 h at 37 °C) or elastase
(1 µg/ml for 2 h at 37 °C), as shown by
immunoprecipitation using anti-LTBP1 and anti-LAP antibodies.
a, autoradiograph showing cleaved LTBP1 (black
arrowhead) and large latent TGF- complex containing cleaved
LTBP1 (white arrowhead) immunoprecipitated from plasmin
(PL) and elastase (EL) digests of
35S-labeled bone ECM. Samples precipitated with antiserum
specific for LTBP1 (Ab) or with control rabbit serum
(C) are indicated above the lanes. b,
autoradiographs showing cleaved LTBP1 (black arrowheads) and
large latent TGF- complex (white arrowhead)
immunoprecipitated from plasmin digests of 35S-labeled bone
ECM (0.1 unit/ml plasmin for 2 h at 37 °C). Samples were
precipitated with anti-LTBP1 or anti-LAP to confirm co-migration. The
controls for these antibodies are normal rabbit serum (RS)
and normal goat immunoglobulin (Gt IgG), respectively.
Molecular mass markers (kDa) are indicated on the right of
the gels. c and d, histograms showing TGF-
concentrations in plasmin or elastase digests of bone ECM, as measured
by bioassay; c, latent TGF-; d, active
TGF-. Samples are as follows: C, control without
proteases; PL, plasmin (0.1 unit/ml for 2 h at
37 °C), EL, elastase (1 µg/ml for 2 h at
37 °C). Note that plasmin releases 15-fold more latent TGF- than
active and that elastase releases only latent TGF-, with
undetectable levels of active TGF-. Values are the means ± S.E. from triplicate samples. The dashed line indicates the
detection limit of the assay. *, significantly different from control
(p < 0.05) using analysis of variance, followed by
Student Newman Keuls method of multiple comparisons.
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To determine whether plasmin and elastase released both latent and
active TGF- from bone ECM, the TGF- concentrations in the plasmin
and elastase digests were measured by bioassay (see Fig. 2,
c and d). Both plasmin and elastase released
latent TGF- from bone ECM (i.e. detectable after acid
activation). Plasmin also released detectable levels of active TGF-.
However, this active TGF- represented less than 10% of the total
TGF- released. In contrast to plasmin, elastase did not release any
detectable active TGF- from bone ECM (Fig. 2d). The doses
of plasmin and elastase used are sufficient to abolish completely the
LTBP1 fibrillar staining in bone matrix preparations from fetal rat
calvarial cells (9).2
To analyze further the production of LTBP1-bound forms of latent
TGF- in primary bone cell cultures, pulse-chase immunoprecipitation experiments were performed (see Fig. 3).
Immunoprecipitation analysis, using a 30-min pulse of
[35S]cysteine labeling, indicated that significant
amounts of LTBP1 were secreted into the culture media of FRC cells
after only 15 min of chase (Fig. 3a). A major band at
170-190 kDa was observed (black arrowhead), corresponding
to free LTBP1. This band represented about 90% of the
immunoprecipitable LTBP1. In addition a minor band at ~290 kDa was
observed (white arrowhead), representing LTBP1 complexed to
TGF-. These bands increased in intensity up to 6 h. However, by
24 and 48 h an additional lower molecular weight band was evident
at ~100 kDa (gray arrow), which presumably represents a
proteolytically processed form of LTBP1.
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Fig. 3.
Pulse-chase immunoprecipitation of LTBP1 in
FRC cells showing rapid secretion of LTBP1 (black
arrowheads) and LTBP1 complexed to TGF-
(white arrowheads) and rapid incorporation into
the ECM. Cells were labeled with [35S]cysteine for
30 min and chased with cold cysteine for the indicated times.
a, LTBP1 immunoprecipitated from the culture media;
b, corresponding control samples precipitated with
non-immune serum; c, LTBP1 immunoprecipitated from plasmin
digests of the extracellular matrix; and d, corresponding
controls precipitated with non-immune serum.
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To detect LTBP1 that is cross-linked in the ECM, the matrix was
digested with plasmin (see above). This resulted in release of
proteolytic fragments of free LTBP1 of 110-130 kDa (Fig. 3c, black arrowheads), and a band representing cleaved LTBP1 complexed to TGF- at 230 kDa (Fig. 3c, white arrowhead).
These experiments indicated that not only was LTBP1 rapidly secreted
but it was also rapidly incorporated into the matrix, with significant
amounts of free LTBP1 and large latent complex detected in the ECM
after only 15 min of chase. Again the intensity of these bands
increased up to 6 h in culture, after which no significant further
increase was observed.
Release of LTBP1 and LTBP1 Complexed with Latent TGF- by Rabbit
Marrow Cells--
To determine whether proteolytic cleavage of LTBP1
could be a potential cellular mechanism for release of matrix-bound
latent TGF-, experiments were performed using cultures of neonatal
rabbit marrow cells. These cultures consist of a mixed population of cells that is highly enriched for mature osteoclasts but also contain
some stromal cells and cells in the osteoblastic lineage, which can
support and activate the osteoclasts. When neonatal rabbit marrow cells
were cultured on [35S]cysteine-labeled bone ECM, specific
radiolabeled products were released into the culture medium, which
could be immunoprecipitated with antibodies against LTBP1. Fig.
4, a and b, shows
radiolabeled fragments released into the culture medium during 0-24 h
and 24-48 h of culture. In control cultures (NC) in which no
cells were seeded onto the labeled matrix, a very small amount of LTBP1
was detected in the medium, presumably representing passive diffusion of LTBP1 from the ECM and/or low level degradation by serum components in the medium. In contrast, when rabbit marrow cells were cultured on
the labeled ECM (C), a major band of ~130 kDa, corresponding to a
cleaved fragment of LTBP1 (black arrowhead), and a minor band of ~230 kDa, corresponding to cleaved LTBP1 complexed to small
latent TGF- (white arrowhead), were released into the
culture medium. Release was approximately equivalent during the 0-24- and 24-48-h culture periods. Treatment with PTHrP, a stimulator of
osteoclast activity through its action on osteoblasts and stromal cells, produced a slight stimulation in release of LTBP1 and LTBP1 complexed to latent TGF-, which was most evident in the first 24-h
culture period. The serine protease inhibitor, aprotinin (AP)
completely blocked release of LTBP1 under both control and PTHrP-stimulated conditions. As expected, release of LTBP1 and LTBP1/TGF- by rabbit marrow cells into the culture medium was concomitant with a reduction in LTBP1 bound in the matrix (see Fig.
4c).
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Fig. 4.
Release of LTBP1 and large latent
TGF- complex from 35S-labeled bone
ECM by rabbit marrow cells as demonstrated by immunoprecipitation using
anti-LTBP1 antibodies. The samples are as follows: NC,
control with no cells; C, rabbit marrow cells;
PTHrP, rabbit marrow cells treated with 100 ng/ml PTHrP;
AP, rabbit marrow cells treated with 50 µg/ml aprotinin;
PTHrP + AP, rabbit marrow cells treated with 100 ng/ml PTHrP
and 50 µg/ml aprotinin. Gels a-c show samples
immunoprecipitated with antiserum specific for LTBP1, and gels
d-f show corresponding control samples immunoprecipitated with
non-immune rabbit serum. a shows cleaved LTBP1 (black
arrowhead) and large latent TGF- complex (white
arrowhead) released into the culture medium by rabbit marrow cells
during 0-24 h of culture. b shows cleaved LTBP1 and large
latent TGF- complex released into the culture medium during 24-48 h
of culture. c shows LTBP1 and large latent TGF- complex
bound in the matrix at the end of the 48-h culture period (the matrix
was treated with plasmin to release the bound LTBP1 and LTBP1/TGF-,
hence a doublet is seen for free LTBP1, see black
arrowheads). Note the release of LTBP1 into the media by rabbit
marrow cells with a corresponding decrease in matrix-bound LTBP1. This
release is blocked by aprotinin.
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Osteoclasts but Not Osteoblasts Release LTBP1 from Bone
ECM--
To rule out that osteoblasts and/or stromal cells in the
rabbit marrow cultures were releasing LTBP1 from bone ECM and to verify
that osteoclasts were responsible for release of LTBP1 and large latent
TGF- complex, experiments were performed using pure populations of
osteoblast-like cells followed by the use of relatively pure
populations of osteoclasts derived from avian osteoclast precursors.
Primary cultures of fetal rat calvarial osteoblasts as well as the rat
osteosarcoma cell line, UMR-106, failed to release LTBP1 and large
latent TGF- complex from bone matrix, either under control or
PTHrP-stimulated conditions (data not shown).
In contrast, when avian osteoclast precursors were cultured on
radiolabeled bone ECM, cleaved fragments of LTBP1 were released into
the culture medium (see Fig. 5,
a-d). These avian osteoclast precursors fuse in culture
over a 6-day period to form highly purified populations of
multinucleated cells (>95% pure), which are positive for
tartrate-resistant acid phosphatase (TRAP) and are capable of resorbing
bone (8, 23). The avian osteoclast cultures released LTBP1
under control, unstimulated conditions (C) (see Fig. 5a, black
arrowheads). This release occurred at a low level throughout the
entire 6-day culture period and was most evident as a dramatic decrease
in the amount of ECM-bound LTBP1 and LTBP1/TGF- at the end of the
culture period (Fig. 5b). Treatment of the avian osteoclast
cultures with 1,25-D3, which enhances fusion of precursors
and stimulates osteoclastic activity (23), resulted in a marked
stimulation in release of LTBP1 and LTBP1/TGF- from bone ECM.
Release was maximal between days 3 and 4 of culture and resulted in the
release of essentially all the ECM-bound LTBP1 and LTBP1/TGF- into
the culture medium by the end of the 6-day culture period (Fig.
5b). For subsequent experiments, the media samples were
therefore collected over days 1-4 of culture. Fig. 5, e and
f, shows photomicrographs of the avian osteoclasts formed
after 6 days of culture on bone ECM under control culture conditions
(Fig. 5e) and in the presence of 1,25-D3 (Fig.
5f). Note that in the presence of 1,25-D3,
fusion of the precursors to form multinucleated osteoclasts is
enhanced, and TRAP staining is much more intense.
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Fig. 5.
Immunoprecipitation showing the time course
of release of LTBP1 and large latent TGF-
complex from 35S-labeled bone ECM by avian
osteoclasts (a-d). Gel a shows
cleaved LTBP1 (black arrowhead) and large latent TGF-
complex (white arrowhead) released into the culture medium
during days 1-2, 3-4, and 5-6 of culture. b shows LTBP1
and large latent TGF- complex bound in the matrix at the end of the
6-day culture period. Gels c and d show
corresponding control samples immunoprecipitated with non-immune serum.
The samples are as follows: NC, control without cells;
C, avian osteoclasts; D3, avian
osteoclasts treated with 10 8 M
1,25-D3. Note a small amount of release of LTBP1 by avian
osteoclasts under control conditions, which is dramatically stimulated
by treatment with 1,25-D3. Maximal release occurs between
days 3 and 4. Release of LTBP1 into the culture medium is associated
with a corresponding decrease in ECM-bound LTBP1 (b). The
photomicrographs in e and f show avian
osteoclasts stained for TRAP activity on day 6 of culture on bone ECM.
Note that under control culture conditions (e), avian
osteoclast precursors fuse to form multinucleated cells but express low
TRAP activity. Treatment with 108 M
1,25-D3 (f) enhances the formation of large
multinucleated cells that strongly express TRAP. Bar, 100 µM. The histogram in g shows the TGF-
content of bone ECM after culture with avian osteoclasts for 6 days.
Control, ECM cultured without avian osteoclasts;
Ocl, ECM cultured with avian osteoclasts; OCl + 1,25-D3, ECM cultured with avian osteoclasts treated
with 108 M 1,25-D3. Data are
expressed as ng TGF- per well and represent the means ± S.E.
of four replicate wells. *, significantly different from ECM alone
(p < 0.05) using analysis of variance, followed by
Student Newman Keuls method of multiple comparisons. h-k,
immunoprecipitation showing the dose-response effects of
1,25-D3 (1010 to 107
M) on release of LTBP1 and LTBP1/TGF- from
35S-labeled bone ECM by avian osteoclasts. The samples are
as follows: NC, control without avian osteoclasts;
C, avian osteoclasts; 10, 9,
8, and 7, avian osteoclasts treated with
1,25-D3 at 1010, 109,
108, and 107 M, respectively.
Gels h and i show samples immunoprecipitated with
antiserum specific for LTBP1, and gels j and k
show corresponding control samples immunoprecipitated with non-immune
rabbit serum. h shows cleaved LTBP1 (black
arrowhead) and large latent TGF- complex (white
arrowhead) released into the culture medium by avian osteoclast
precursors during days 1-4 of culture. i shows LTBP1
(black arrowhead) and large latent TGF- complex
(white arrowhead) bound in the matrix at the end of the
culture period. Note that concentrations as low as 1010
M 1,25-D3 stimulated release of LTBP1 by avian
osteoclasts. Maximal release was observed with
108-107 M.
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To confirm that the avian osteoclasts were releasing TGF- from the
bone matrix, the TGF- content of the bone ECM was measured after
culturing with avian osteoclasts for 6 days (see Fig. 5g). Bone ECM that had been cultured with avian osteoclasts showed an
~60% reduction in TGF- content compared with controls cultured without cells. Stimulation of the avian osteoclasts with
108 M 1,25-D3 further enhanced
this release, resulting in the loss of ~90% of the matrix-bound
TGF-.
Dose-response experiments indicated a dramatic stimulation in release
of ECM-bound LTBP1 by avian osteoclast precursors treated with doses of
1,25-D3 as low as 1010 M compared
with unstimulated controls (see Fig. 5, h and i). Maximal release was seen between 108 and
107 M and subsequent experiments were
performed using the 108 M dose of
1,25-D3.
Effects of Protease Inhibitors on Release of Bone Matrix-bound
LTBP1 and LTBP1 Complexed to Latent TGF- by Avian
Osteoclasts--
The osteoclast culture systems we have used produce
complex mixtures of proteases including cathepsins, plasminogen
activators, and matrix metalloproteinases (MMPs) (reviewed in Ref. 31). In particular, osteoclasts express high levels of cathepsin K (32) and
MMP9 (33, 34). To investigate which protease(s) were responsible for
cleavage of LTBP1, a number of protease inhibitors were examined (see
Fig. 6). Under basal, unstimulated
conditions (C), only a small amount of LTBP1 was released from
radiolabeled bone ECM by avian osteoclasts when compared with matrix
cultured without cells (NC). This low level release was blocked by the serine protease inhibitor, AP. The other protease inhibitors tested did
not have any notable effect on basal release of LTBP1/TGF- from bone
ECM. These included the serine protease inhibitor leupeptin, the
cysteine protease inhibitor pepstatin A, and the tissue inhibitor of
matrix metalloproteinases-1 (TIMP1). In contrast, under
1,25-D3-stimulated conditions, essentially all the
matrix-bound LTBP1 and large latent TGF- complex were released into
the culture media. Both the serine protease inhibitor, aprotinin, and
the matrix metalloproteinase inhibitor, TIMP1, blocked release of LTBP1
and LTBP1/TGF- almost to the level seen in unstimulated control
cells. The serine protease inhibitor leupeptin also
partially blocked release of LTBP1 and LTBP1/TGF-. In contrast, the
aspartic (acid) protease inhibitor, pepstatin A, was without effect. In
separate experiments we have also found that the cysteine protease
inhibitor E64 is without effect in this assay (data not shown).
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Fig. 6.
Immunoprecipitation showing release of LTBP1
(black arrowhead) and large latent
TGF- (white arrowhead) from
[35S]cysteine-labeled bone ECM by avian osteoclasts and
inhibition by protease inhibitors. a shows release of LTBP1
and large latent TGF- complex into the culture medium over the 4-day
culture period; b shows LTBP1 and large latent TGF-
complex bound in the ECM at the end of the 4-day culture. Samples are
as follows: NC, control without osteoclasts; C,
avian osteoclasts; AP, avian osteoclasts treated with
aprotinin (50 µg/ml); leu, avian osteoclasts treated with
leupeptin (10 µM); pep, avian osteoclasts
treated with pepstatin A (1 µM); TIMP, avian
osteoclasts treated with TIMP-1 (1.5 µg/ml). Samples treated with
108 M 1,25-D3 are denoted
+D3. Note the release of LTBP1 and LTBP1/TGF-
by avian osteoclasts stimulated with 1,25-D3. Release was
blocked to control levels using inhibitors of serine proteases
(aprotinin and leupeptin) and by an inhibitor of MMPs (TIMP-1) but not
by an inhibitor of aspartic (acid) proteases (pepstatin A).
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Cleavage of LTBP1 by Purified MMPs--
The above experiments
suggested the involvement of serine proteases and/or matrix
metalloproteinases in release of LTBP1 and large latent TGF- complex
from bone ECM by avian osteoclasts. To confirm that MMPs were able to
cleave LTBP1, experiments were performed using the purified proteases.
LTBP1 was immunoprecipitated from the conditioned media of CHO-L76
cells which stably overexpressed human LTBP1 using a mouse monoclonal
antibody against LTBP1. The immunoprecipitates were then digested with
plasmin, MMP2, or MMP9 and analyzed by immunoblotting using a rabbit
polyclonal antibody against LTBP1 (see Fig.
7a). LTBP1 immunoprecipitates
incubated with digestion buffer alone (C) gave a single LTBP1
immunoreactive band at ~190 kDa (black arrow). Digestion
with plasmin (PL), MMP2, or MMP9 produced degradation products in the
range 125-165 kDa (gray arrows), confirming cleavage
of LTBP1. As expected, no immunoreactive LTBP1 bands were detected in
samples precipitated with control IgG.
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Fig. 7.
a, Western blot showing cleavage of
LTBP1 by plasmin (PL), MMP2, and MMP9. LTBP1 was
immunoprecipitated from the conditioned media of CHO-L76 cells stably
transfected with human LTBP1, using a monoclonal antibody specific for
human LTBP1. The immunoprecipitate was digested with plasmin, MMP2, or
MMP9 (25 µg/ml) for 6 h at 37 °C and then electrophoresed in
4-20% gradient SDS-PAGE gels under non-reducing conditions. The
samples were immunoblotted using a polyclonal antibody to LTBP1 (Ab39).
The samples are as follows: IgG, sample immunoprecipitated
with non-immune IgG; C, sample immunoprecipitated with LTBP1
monoclonal antibody and incubated in digestion buffer alone;
PL, MMP2, and MMP9, sample
immunoprecipitated with anti-LTBP1 monoclonal antibody and then
digested with the indicated protease. Note cleavage of
immunoprecipitated LTBP1 by plasmin, MMP2, and MMP9 to give fragments
in the range 125-165 kDa. b, Western blot showing LTBP1
fragments released by digestion of ECM from fetal rat calvarial cells
with plasmin (PL), MMP2, and MMP9. ECM preparations were
digested with plasmin, MMP2, or MMP9 (25 µg/ml) for 6 h at
37 °C and then electrophoresed in 4-20% gradient SDS-PAGE gels
under non-reducing conditions. The samples were immunoblotted using a
polyclonal antibody to LTBP1 (Ab39). Note that plasmin and MMP2
released cleaved LTBP1 fragments; however, MMP9 failed to release LTBP1
fragments from the ECM. c, immunofluorescent images of ECM
from fetal rat calvarial cells following digestion with PL, MMP2, and
MMP9. Note the well organized fibrillar network in control cultures
(C), the reduced staining intensity following MMP2
digestion, and virtual absence of fibrillar staining following plasmin
treatment. MMP9 treatment failed to reduce the fibrillar staining
significantly compared with undigested controls (bar, 25 µm).
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We also examined the ability of MMPs to cleave LTBP1 in its ECM-bound
form, which is localized to 10 nm matrix microfibrils in primary
osteoblast cultures (10). Treatment of osteoblast-derived ECM with
either plasmin or MMP2 resulted in the release of cleaved fragments of
LTBP1 into the digest supernatant in the range 125-150 kDa (see Fig.
7b). In contrast, MMP9 failed to release significant amounts
of LTBP1 from the ECM. Activity of the MMP2 and MMP9 preparations was
confirmed by gelatin zymogram analysis (data not shown). Western blot
data were confirmed by parallel immunofluorescent staining for LTBP1 in
matrix preparations that were digested with plasmin, MMP2, or MMP9 (see
Fig. 7c). Fibrillar LTBP1 staining was observed in control
cultures (C). Treatment with MMP2 dramatically reduced fibrillar LTBP1
immunostaining, and treatment with PL almost completely abolished LTBP1
staining. In contrast, treatment with MMP9 produced only a slight
reduction in staining intensity.
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DISCUSSION |
Although LTBP1 was originally identified as a component of the
large latent TGF- complex (13, 14), it is now clear that the LTBPs
1-4 are members of the fibrillin superfamily that have multiple functions as both structural ECM proteins and as regulators of
TGF- availability (11, 12). Our data, showing release of ECM-bound
LTBP1 and LTBP1 complexed to latent TGF- by osteoclasts, represent
the first demonstration that proteolytic cleavage of LTBP1 may be a
physiological mechanism for the release of latent TGF- from
ECM-bound stores. Although previous studies (9, 20, 21) using purified
proteases have implied a proteolytic mechanism for release of latent
TGF- from ECM through cleavage of LTBP1, the amounts of proteases
used in these studies are high relative to physiological
concentrations. Our studies using osteoclast populations show that
these cells, which are actively involved in matrix turnover, are able
to release cleaved fragments of LTBP1 and LTBP1 complexed to latent
TGF- from matrix that is laid down by osteoblasts.
In the present studies we have also characterized the forms of latent
TGF- produced by primary osteoblasts and incorporated into the ECM.
The major secreted forms were the small latent complex containing
TGF-1 and large latent complexes containing predominantly TGF-1,
with smaller amounts of TGF-2. Previously, we reported characterization of the forms of latent TGF- produced by
osteosarcoma cell lines (24). Whereas one cell line (UMR-106) produced
exclusively the small latent TGF- complex, and one (MG63) produced
exclusively the large latent TGF- complex, a third cell line (ROS
17/2.8) produced both complexes and therefore resembled most closely
the primary bone cells examined in the present studies. Pulse-chase immunoprecipitation studies demonstrated that the large latent complex
containing LTBP1 is rapidly secreted and rapidly incorporated into the
ECM of primary osteoblasts, similar to data reported using a human
fibrosarcoma cell line (19). However, in contrast to the fibrosarcoma
cells, the major proportion of the LTBP1 produced by primary
osteoblasts remained in the culture medium. This suggests that either
the osteoblast cells produce an excess of LTBP1 over that which is
required for matrix incorporation or that the mechanisms controlling
matrix incorporation may be different in these two cell types.
Interestingly, only a minor fraction (~10%) of the LTBP1 produced by
primary osteoblasts was complexed to TGF-, with the remaining 90%
occurring in a free, uncomplexed form. In various cell types examined,
including fibroblasts (19, 24), osteoblasts, (9, 24) and breast cancer
cells,2 the amount of free LTBP1 can vary between 50 and
95%. This suggests functions for LTBP1 that are independent of its
role in TGF- regulation. In previous studies (9, 10) we have
demonstrated that bone matrix-bound LTBP1 is present as part of an
organized microfibrillar network, which also contains fibrillin-1.
These observations, together with the high degree of homology of LTBP1 with the fibrillins, support an important independent role for LTBP1 as
an extracellular matrix protein.
Release of bone matrix-bound TGF- by resorbing osteoclasts would be
expected to have important consequences for bone cells, as TGF- is
capable of regulating all the steps in the bone remodeling cascade
(reviewed in Ref. 5). TGF- has been shown to inhibit osteoclast
activity, both by stimulating osteoclasts to undergo apoptosis and by
inhibiting formation of osteoclasts from their precursors. TGF- is
also a powerful chemoattractant and mitogen for osteoblast precursors
(5). Therefore, TGF- released from bone matrix could attract
osteoblasts to the site of bone resorption and stimulate them to
proliferate. The effect of TGF- on mature osteoblasts is then to
inhibit proliferation and stimulate production of bone ECM proteins,
including type I collagen, fibronectin, and osteocalcin (reviewed in
Ref. 35). In addition to stimulating these ECM proteins, TGF-1 also
induces expression of its own message as well as expression of LTBP1
(24, 35).
Interestingly, 1,25-dihydroxyvitamin D3 stimulated release
of LTBP1 from bone ECM by avian osteoclast cells. This effect is most
likely due to 1,25-D3 enhancing resorptive activity through stimulating fusion of osteoclast precursors to form mature resorbing cells. However, a direct effect of 1,25-D3 on protease
activity in osteoclasts is also possible.
Our studies using protease inhibitors suggested the involvement of
serine proteases and/or MMPs in release of ECM-bound LTBP1 and LTBP1
complexed to latent TGF- by avian osteoclasts and rabbit bone marrow
cells. These findings are consistent with our biochemical observations
that plasmin, MMP2, and MMP9 can cleave LTBP1. Our data using
inhibitors of cysteine and aspartic proteases (e.g. cathepsins B, L, D, and K) suggest that this group of proteases are not
involved in cleavage of LTBP1 by avian osteoclasts. This is in
agreement with the biochemical data of Taipale and co-workers (21)
showing that cathepsins B and D were negative or showed a very limited
ability to cleave LTBP1 from the ECM of epithelial cells.
Our studies identify LTBP1 as a new substrate for MMPs. Interestingly,
both MMP2 and MMP9 were able to cleave the soluble form of LTBP1;
however, only MMP2 cleaved the ECM-bound form of LTBP1. This suggests
that the cleavage sites used by MMP9 may be unavailable when LTBP1 is
incorporated into the ECM. Thus, efficient cleavage of soluble forms of
LTBP1 by a protease may not necessarily imply that the same protease
will cleave LTBP1 once it is assembled into ECM microfibrils. Another
possibility is that degradation of other collagenous or non-collagenous
ECM components may be required prior to LTBP1 cleavage to expose
cryptic proteolytic cleavage sites on LTBP1. Thus, a cascade of
proteases that are involved in the degradation of bone ECM by
osteoclasts may be required for release of LTBP1 and TGF- from bone
ECM.
Recent studies (36, 37) have shown that other members of the fibrillin
superfamily, such as fibrillins 1 and 2, are also substrates for MMPs.
Breakdown of microfibrillar proteins by MMPs may be important in
pathological conditions and may contribute to the disease phenotype in
inherited disorders such as Marfan's and related syndromes.
Interestingly, fibrillin-1 mutations associated with Marfan's syndrome
and ectopia lentis have been shown to result in the production of
mutant protein, which is more susceptible to degradation by proteases
including MMPs (36, 37). This effect appears to be through disruption
of calcium binding and apparent exposure of cryptic protease cleavage
sites. Breakdown of fibrillin-containing microfibrils that also contain
LTBP1 would be expected to result in the release and activation of
matrix-bound TGF-, which has been strongly implicated in a number of
fibrotic diseases and could play a role in scleroderma, another disease for which the fibrillin-1 gene has been implicated (reviewed
in Ref. 38).
Our studies showing release of TGF- from bone matrix by plasmin and
elastase suggest that these proteases are much more efficient at
cleaving LTBP1 and releasing latent TGF- from bone matrix than they
are at activating the latent TGF- released. Thus, concentrations of
plasmin that can cleave essentially all the LTBP1 and abolish LTBP1
immunoreactivity in the ECM are capable of activating only a small
proportion of the latent TGF- released. Although several studies
(29, 30) have implicated plasmin as an activator of TGF-, these
studies have been performed using conditioned medium containing
predominantly the 100-kDa small latent TGF- complex, which may be
more readily activated than the large latent complex, or samples in
which the latent TGF- complexes are not defined. Other studies
(reviewed in Ref. 39) have been performed using co-culture systems,
where again the form of latent TGF- is not defined and may involve
release from ECM-bound stores. Our data support the notion that there
may be several sequential steps in the process by which ECM-bound
TGF- is rendered active, as suggested by Rifkin and co-workers (40).
Proteases such as plasmin and MMPs may be important initially in
release of latent TGF- from ECM-bound stores. Activation may then
take place by cell surface-localized mechanisms, perhaps involving
interactions with mannose 6-phosphate receptors (40), integrins (41),
or thrombospondins (42). A recent report (43) has also implicated
surface-bound forms of MMP2 and MMP9 in activation of the small latent
TGF- complex. Further studies are therefore clearly warranted to
clarify the steps in the activation pathway.
This study has focused on one potential pathway for release of latent
TGF- from bone ECM. However, other mechanisms for TGF- storage
and release in bone ECM may also play an important role. For instance,
many growth factors can bind to matrix via heparan sulfate-containing
proteoglycans and other members of the proteoglycan family. Mature
TGF- can bind to heparin (44) and our own unpublished studies
indicate that LTBP1 itself binds to
heparin.3 Studies by
Tiedemann et al. (45) have demonstrated the importance of
heparan sulfate-containing proteoglycans in assembly of fibrillin-1 into the ECM. Thus heparan sulfate-containing proteoglycans may play a similar role in LTBP1 (and by implication TGF-)
incorporation. At present the potential role of proteoglycan-degrading
enzymes, such as heparanase, in release of TGF- from bone ECM
remains to be determined. However, Taipale et al. (21)
reported that various glycosidases, including heparinases I and III and
chondroitinase ABC as well as a combination of all three, were unable
to cleave LTBP1 from the ECM of epithelial cells.
Mature TGF- is also known to bind to proteoglycans such as decorin
and biglycan, which may provide an alternative pathway for storage and
release of TGF- in bone ECM (46). These latent TGF- complexes
could be viewed as "secondary" complexes because it is the mature
TGF- homodimer (i.e. after activation) that is bound. In
contrast, the LTBP1-bound TGF- can be thought of as a "primary"
latent TGF- complex, because this latent complex is assembled inside
the cell prior to secretion (17). Although our studies have
demonstrated a likely role for LTBP1 in both the storage and release of
latent TGF- in bone ECM, other secondary latent TGF- complexes,
such as the decorin and biglycan-bound forms, may also be released and
activated during bone resorption.
In summary, the data presented here, using osteoclast culture systems,
provide the first demonstration that cell-mediated release of ECM-bound
TGF- can occur via proteolytic cleavage of LTBP1. LTBP1 is known to
regulate TGF- activity at multiple levels including enhancing
secretion of the latent TGF- complex, facilitating storage of the
latent TGF- complex in the ECM and modulating activation of latent
TGF-. The involvement of LTBP1 in the cell-mediated release of
latent TGF- from ECM-bound stores confirms an additional regulatory
role for this ECM protein and may provide an important pathway for
communication between cells and the extracellular matrix.
(TGF-
§¶,
TOP
ABSTRACT
INTRODUCTION
