(Received for publication, October 3, 1994; and in revised form, November 17, 1994)
From the
Monolayer cultures of human epithelial and endothelial cells
were used to study the association of latent transforming growth
factor-1 (TGF-
1) to extracellular matrices and its release
and activation during matrix degradation. Human umbilical vein
endothelial cells and embryonic lung fibroblasts produced relatively
high levels of TGF-
1, its propeptide (
1-latency-associated
protein), and latent TGF-
-binding protein and incorporated latent
TGF-
1 into their matrices as shown by immunoblotting. Amnion
epithelial cells produced lower levels of these proteins. Confluent
cultures of epithelial cells were exposed to matrix-degrading proteases
and glycosidases. Mast cell chymase, leukocyte elastase, and plasmin
efficiently released matrix-bound latent TGF-
1 complexes, while
chondroitinase ABC and heparitinases were ineffective. The ability of
the proteases to activate recombinant latent TGF-
1 was tested
using growth inhibition assays and a novel sodium
deoxycholate-polyacrylamide gel electrophoresis followed by
immunoblotting. Sodium deoxycholate solubilized M
25,000 TGF-
1 but did not dissociate high M
latent TGF-
1 complexes, allowing separation of these forms
by polyacrylamide gel electrophoresis. Mast cell chymase and leukocyte
elastase did not activate latent TGF-
1, suggesting that its
release from matrix and activation are controlled by different
mechanisms. The release of TGF-
from the matrix by leukocyte and
mast cell enzymes may contribute to the accumulation of connective
tissue in inflammation.
Transforming growth factor-s are a family of
multifunctional polypeptide growth factors/growth
inhibitors(1, 2, 3, 4, 5, 6, 7) .
Three isoforms of TGF-
(
)have been found in mammals
(TGF-
1-TGF-
3). TGF-
s are commonly secreted by
cultured cells in a latent form(8) . Latency is caused by the
amino-terminal pro-domain (LAP, latency-associated protein), which is
cleaved from carboxyl-terminal active TGF-
during secretion but
remains non-covalently associated to the mature dimer(9) . The
latent complex consisting of
1-LAP and TGF-
1 has been named
small latent TGF-
1(10) . A major fraction of latent
TGF-
from a variety of cell lines forms large latent TGF-
complexes, containing additional high molecular mass proteins that
associate with LAP(10, 11, 12, 13) .
Best characterized of these is the latent TGF-
-binding protein
(LTBP), which associates to LAP by a disulfide
bond(11, 14, 15, 16) . LTBP is
important in the processing and secretion of latent
TGF-
(12) . We have recently observed that LTBP and large
latent TGF-
1 are extracellular matrix proteins forming insoluble
high molecular mass complexes(13, 17) . These
cross-linked forms can be solubilized by plasmin, which cleaves LTBP at
specific sites, releasing both free LTBP and LTBP complexed to
LAP
TGF-
1 to the culture medium.
LTBP shows extensive
homology in domain structure to fibrillins, a group of microfibrillar
extracellular matrix
proteins(14, 18, 19, 20) . LTBP
localizes to the extracellular matrix of normal tissue and cancer
stroma(20) . Staining of LTBP often parallels collagen
fibers(20, 21, 22) . TGF-s also often
colocalize with interstitial extracellular matrix components and
basement membranes(23, 24, 25) . In addition,
soluble latent or active TGF-
is commonly absent from the
supernatant fluids of homogenized tissues(22) , unless
extracted by acid/ethanol or denaturants(26, 27) .
Since most tissues and cultured cells express different isoforms of
TGF- and its receptors(1, 6, 25) , the
events that activate latent TGF-
are probably the major mechanisms
controlling TGF-
activity in tissues. In vitro, latent
forms of TGF-
can be activated by extremes of pH, by heat
treatment, and by certain glycosidases and the protease
plasmin(8, 28, 29, 30, 31, 32, 33, 34) .
Proteolysis (29, 30, 31, 32, 33, 34) ,
acidic cellular microenvironments(35) , and the extracellular
matrix molecule thrombospondin (36) have been proposed to
mediate TGF-
activation under physiological conditions.
TGF- enhances the formation of connective tissue by stimulating
the proliferation of matrix-producing mesenchymal cells such as smooth
muscle cells and fibroblasts via an autocrine platelet-derived growth
factor loop(37) , and by increasing the production of
extracellular matrix components(1, 5, 38) .
Connective tissue growth is further enhanced by the inhibition of
epithelial and endothelial cell proliferation (39, 40) and decrease in matrix degradation by
induction of proteinase inhibitors, such as plasminogen activator
inhibitor-1 and tissue inhibitor of
metalloproteinases-1(41, 42) . TGF-
promotes
tissue fibrosis and inflammatory cell infiltration by the induction of
fibroblast, monocyte, and neutrophil
chemotaxis(43, 44, 45) . On the other hand,
TGF-
is immunosuppressive, and mice lacking both TGF-
1
alleles die shortly after weaning of excessive inflammatory response (46) .
These properties indicate that TGF- activity is
important for the healing of wounds and damaged
tissues(4, 47, 48) . Disturbances in the
activation of TGF-
may, in turn, contribute to fibrotic responses
in a variety of diseases, such as glomerulonephritis(4) , lung
fibrosis (49) , liver cirrhosis(50) , fibrosis of
transplanted tissues during allograft rejection(22) , and in
fibrosis of arteries after coronary angioplasty(51) , in
carcinoid heart disease (21) and in
arteriosclerosis(52) .
The present study was undertaken to
understand the production and matrix deposition of latent TGF-1
complexes in two monolayer cell systems, namely cultured human
epithelial and endothelial cells, and the roles of two important
enzymes, human leukocyte elastase and mast cell chymase, on the
TGF-
1-containing extracellular matrices.
Figure 1:
Analysis of latent forms of TGF-1
from epithelial and endothelial cells by immunoblotting. After reaching
confluence, cultures of human amniotic epithelial cells (Ep),
umbilical vein endothelial cells (En), and embryonic lung
fibroblasts (Fb) were washed and changed to serum-free medium,
and conditioned medium was harvested at 96 h. A, secretion of
LTBP,
1-LAP, and TGF-
1 to the culture medium. Samples of the
conditioned medium (30 µl, equal to proteins secreted by 0.2
cm
of cells) were analyzed by 4-15% gradient SDS-PAGE
(no stacking gel) under non-reducing conditions followed by
immunoblotting with antibodies specific for LTBP,
1-LAP, and
TGF-
1 as indicated. Lanes denoted LTBP and LL-TGF
1 contain 1 ng of recombinant human free form of
LTBP and large latent TGF-
1, respectively. Migration of the
molecular mass markers (kDa) are shown on the left. B, analysis of TGF-
1 complexes by sodium
deoxycholate-PAGE. The panel shows the immunoblotting analysis for
TGF-
1 of the respective samples run in 4-15% gradient sodium
deoxycholate-PAGE (40 µl of samples concentrated 20-fold by
ultrafiltration; equal to proteins secreted by 5 cm
of
cells). TGF-
1 standard lanes L, S, and A contain 4 ng of large latent TGF-
1 (L, 400 pg of
TGF-
equivalent), 10 ng of small latent TGF-
(S, 2.5
ng of TGF-
equivalent), and 700 pg of active TGF-
1 (A), respectively. Activation control lanes contain fibroblast
conditioned medium treated with medium only(-), acid
(100 mM HCl), or alkali (Alk, 100 mM NaOH) prior to neutralization and sample loading. TGF-
1
standard lanes are from a 10-fold shorter exposure than the first three
lanes for clarity, see panel A for quantitative results. Arrow marks the position of the bottom of the sample wells
(stacking gel was not used). C, analysis of the extracellular
matrices. Extracellular matrices were prepared from confluent cultures
of the respective cells. Immunoblotting analysis on 4-15%
nonreducing gradient SDS-PAGE gels (no stacking gel) were carried out
from samples of matrix (1 cm
/lane) with antibodies specific
for LTBP (upper panel),
1-LAP (middle panel),
and the TGF-
1 (lower panel) after solubilization of the
matrices by SDS(-) or limited plasmin digestion followed by SDS
(+, see ``Materials and Methods''). Lane L contains 1 ng of large latent TGF-
1 complex. The migration of
molecular mass markers (kDa) is indicated on the right.
The molecular complexes of secreted latent TGF- were then
characterized by sodium deoxycholate-PAGE. Sodium deoxycholate is less
denaturing than SDS and does not dissociate TGF-
1 from the small
or large latent complexes(13) . In accordance with the results
presented above, the majority of soluble fibroblast and endothelial
cell-derived TGF-
1 comigrated with recombinant large latent
TGF-
1 (LTBP:LAP
TGF-
1). Approximately 10-20% of
TGF-
1 in endothelial cell and 5% in fibroblast conditioned medium
comigrated with the small latent TGF-
1 (LAP
TGF-
1),
respectively (Fig. 1B). Epithelial cells secreted
significantly lower amounts of TGF-
1 in two different TGF-
1
complexes, the small latent complex (
50%), and a novel large latent
complex migrating above the LTBP:LAP
TGF-
1 (
50%).
Unlike active TGF-1 obtained by heat treatment, which
aggregates on top of the gel(13) , acid-activated TGF-
1
migrated as a low molecular mass band in sodium deoxycholate-PAGE (Fig. 1B). Immunoreactive band comigrating with active
TGF-
1 was not detectable in any cell type, confirming earlier
observations that TGF-
1 secreted by cultured cells is usually in
the latent form.
Latent TGF-1 and LTBP associate with the
extracellular matrices of cultured human fibroblasts and fibrosarcoma
cells(13) . We therefore prepared extracellular matrices of
confluent cultures of amnion epithelial cells, endothelial cells, and
fibroblasts by sodium deoxycholate extraction (see ``Materials and
Methods''). Since the major fraction of LTBP in fibroblast
matrices is cross-linked to high molecular mass complexes that do not
enter SDS-PAGE gels(13) , some matrix preparations were
digested with plasmin (see ``Materials and Methods'') to
unmask LTBP immunoreactivity. Immunoblotting of the plasmin-digested
matrices revealed that matrices of all cell types contained comparable
amounts of free LTBP (approximately 1-3 ng/cm
; Fig. 1C, top panel). The matrices of
endothelial cells and fibroblasts contained significant amounts of
TGF-
1 (30-70 pg/cm
; Fig. 1C, bottompanel) and LTBP:LAP (200-600
pg/cm
; Fig. 1C, middlepanel), whereas in epithelial cell matrices TGF-
1
was barely detectable and LTBP:LAP was below detection limit (Fig. 1C, bottom and middlepanels). In matrices solubilized with SDS alone, multiple
very high molecular mass LTBP immunoreactive bands were detected by
immunoblotting, and some LTBP was aggregated on the top of the gel.
1-LAP was not detectable without plasmin treatment. Endothelial
cell matrices contained two nonspecific background bands (Fig. 1C;
160 and 70 kDa). Small latent TGF-
1
was not detectable in the matrix of any of the cells (Fig. 1C, middlepanel).
Amnion
epithelial cells secreted very low levels of LTBP, while their matrices
contained significant amounts of it. To test the possibility that these
cells did not produce LTBP, but assembled exogenous LTBP from serum to
their matrices, we metabolically labeled amnion epithelial cells with
[S]cysteine. LTBP could be immunoprecipitated
from plasmin solubilized matrix of these cells (see (13) for
details), indicating that it was in part produced by the cells (data
not shown). The efficient incorporation of LTBP to amnion epithelial
cell matrices is probably related to the polar nature of this cell
type.
For the analysis of matrix-degrading enzymes, confluent cultures of amnion epithelial cells were washed, changed to serum-free conditions for 4 h and treated with increasing concentrations of human mast cell chymase and tryptase, leukocyte elastase, plasmin, and cathepsin G, porcine pancreatic elastase, bovine spleen cathepsin B and D, C. histolyticum collagenase, F. heparinum heparinases I and III, and P. vulgaris chondroitinase ABC. Immunoblotting analysis of released material and matrices indicated that mast cell chymase, leukocyte and pancreatic elastases, and plasmin effectively released LTBP from the matrix of the cells ( Table 1and Fig. 2). Low activity was displayed by cathepsin G, mast cell tryptase, cathepsin B, and bacterial collagenase (in this order of potency), while cathepsin D and the glycosaminoglycan-degrading enzymes were negative ( Table 1and Fig. 2).
Figure 2: Release of LTBP from epithelial cell matrix by selected enzymes. Confluent cultures of human amnion epithelial cells were changed to serum-free conditions and incubated with matrix-degrading enzymes for 1 h. Medium was collected and extracellular matrices were prepared from the cells by sodium deoxycholate extraction followed by solubilization by limited plasmin digestion and SDS (see ``Materials and Methods'' for details). LTBP was assayed from the medium (Released) and solubilized extracellular matrix (Matrix) by non-reducing SDS-PAGE followed by immunoblotting (4-15% gradient gel). Chy, human mast cell chymase; PE, porcine pancreatic elastase; LE, human leukocyte elastase; Pla, plasmin; Try, mast cell tryptase; Col, bacterial collagenase; B, D, and G, respective cathepsins; HI and HIII, heparinases I and III; ABC, chondroitinase ABC; All, heparinases I, III, and chondroitinase ABC. Doses used are maximum tested doses of Table 1. The migration of molecular mass markers is indicated on the right.
Mast cell chymase, leukocyte and pancreatic elastases, and plasmin processed LTBP to similar molecular mass forms (90-130 kDa, Fig. 2) suggestive of protease-resistant ``core'' domain. Immunoreactive bands of lower molecular mass were not produced by any of the proteinases tested. The platelet-like LTBP is highly resistant to digestion with proteinases(12, 13, 59) . This was further emphasized by the observation that 100-fold higher concentrations of porcine pancreatic elastase that are sufficient to release all of matrix-bound LTBP could not further degrade the 90-kDa form of LTBP (Fig. 2).
Figure 3:
Release of LTBP and TGF-1 from
endothelial cell matrix by proteinases. Confluent cultures of human
umbilical vein endothelial cells were changed to serum-free conditions
and incubated for 1 h with increasing concentrations of human plasmin,
mast cell chymase, leukocyte elastase, and thrombin. Release of LTBP
and TGF-
1 from the matrix (ECM) to culture supernatant (Released) was assayed by immunoblotting as described in
caption to Fig. 2. In panelsLTBP, lower
molecular mass band(s) represent free LTBP, and higher molecular mass
band(s) LTBP complexed to
-LAP.
Figure 4: Processing of LTBP from fibroblast conditioned medium by selected proteinases. Fibroblast conditioned medium was harvested at 3 days and incubated with proteinases at 37 °C for 1 h. Reactions were terminated by the addition of SDS (2% final concentration), and the samples were analyzed by 4-20% gradient SDS-PAGE under nonreducing conditions followed by immunoblotting with LTBP-specific antibodies. Ct, untreated medium; Pla, plasmin (50 nM); Chy, chymase (10 nM); Leu, leukocyte elastase (10 nM); Pan, porcine pancreatic elastase (10 nM); G, cathepsin G (100 nM); B, cathepsin B (500 nM); D, cathepsin D (500 nM). The migration of molecular mass markers (kDa) is indicated on the right.
It is known
that low concentrations of plasmin (10-100 nM) and
thrombin release TGF-1 from the matrix of fibroblasts and
endothelial cells in a latent
form(17, 60, 61) . We studied next the
bioactivity of TGF-
released from the matrix of endothelial cells
by mast cell chymase, leukocyte elastase, and cathepsin G. Confluent
cultures of endothelial cells were treated with these proteinases for 2
h, and the activity of TGF-
1 released to the culture medium was
determined by Mv1Lu growth inhibition assay. The supernatant fluids
were not growth-inhibitory to Mv1Lu cells, indicating that the samples
contained less than 50 pg/ml active TGF-
(Fig. 5). In
addition, no TGF-
1 activity was detected in the supernatants using
an ELISA that is specific for active TGF-
1 (R& Systems;
detection limit
30 pg/ml; data not shown). Supernatant fluids of
chymase, leukocyte elastase, and plasmin-treated cells were 95% growth
inhibitory to Mv1Lu cells after heat treatment (80 °C, 5 min),
indicating that these samples contained >1 ng/ml latent TGF-
(Fig. 5). These results indicate that TGF-
released by
chymase, elastase, and plasmin was >95% latent, since >1 ng/ml
latent TGF-
and <0.05 ng/ml of active TGF-
was released by
these proteinases.
Figure 5:
Analysis of the biological activity of
released TGF- by Mv1Lu cell growth inhibition assay. Confluent
cultures of human endothelial cells were changed to serum-free
conditions and treated with mast cell chymase (30 nM),
leukocyte elastase (3 nM), and cathepsin G (3 nM) for
2 h at 37 °C. Plasmin (30 nM) and human matrix
metalloproteinase-3 (stromelysin; 100 nM) were used as
positive and negative controls, respectively. Samples of medium
representing 3 cm
of cell layer were diluted to 500 µl
of minimal essential medium and analyzed by Mv1Lu cell growth
inhibition assay in triplicate. Some samples were heat treated (80
°C, 5 min) prior to analysis to activate latent forms of TGF-
.
Growth inhibition was assessed after 18 h by
[6-
H]thymidine incorporation. Standards of active
TGF-
are also indicated.
We studied then the ability of the proteinases to
activate recombinant human large latent TGF-1. Large latent
TGF-
1 (10 µg/ml) was incubated with mast cell chymase (500
nM), leukocyte cathepsin G (500 nM), or elastase (50
nM) for 4 h at 37 °C in the absence or presence of heparin
(10 µg/ml). A sample treated with a high concentration of plasmin
(1 µM) was included as a positive control of
protease-mediated TGF-
activation(29, 30) . The
proteinases were inactivated by the addition of proteinase inhibitors
and TGF-
1 bioactivity was analyzed by Mv1Lu growth inhibition
assay. Large latent TGF-
1 (200 pM) treated with mast cell
chymase or leukocyte elastase was not significantly growth inhibitory
to Mv1Lu cells. Since the detection limit of the assay is
2
pM, this indicates that less than 1% of large latent
TGF-
1 could be activated by these treatments. Approximately
1-2% of plasmin-treated large latent TGF-
1 was activated,
corresponding to 4 pM TGF-
1 (see also (29) and (30) ). Cathepsin G treatment induced marginal activation of
large latent TGF-
1.
The samples were then analyzed by sodium
deoxycholate-PAGE, followed by immunoblotting with TGF-1 specific
antibodies. Treatment of the large latent TGF-
1 with leukocyte
elastase or mast cell chymase did not dissociate TGF-
1 from the
latent complex, while cathepsin G induced a very weak species
comigrating with active TGF-
1. In control samples treated with
plasmin, a clear band comigrating with active TGF-
1 was detected
(representing
1% of total TGF-
1; for pH activation controls,
see Fig. 1B). Similar levels of activation (
1%) of
large latent TGF-
1 by plasmin was also detected by an ELISA that
is specific for active TGF-
1 (data not shown). Leukocyte elastase
treatment converted approximately 50% of large latent TGF-
1 to a
band migrating between small and large latent complexes (Fig. 6, top panel). Analysis of the samples by SDS-PAGE followed by
immunoblotting with LTBP specific antibodies indicated that the change
in migration was caused by degradation of LTBP as indicated by the
decreased LTBP immunoreactivity at 200 kDa (Fig. 6, bottompanel). This may represent an unphysiological effect,
since LTBP released from the matrix was not completely degraded by
leukocyte elastase (see Fig. 2and Fig. 3) and heparin,
its physiological cofactor, totally inhibited the degradation of LTBP (Fig. 6, top and bottompanels).
Other proteinases did not result in significant changes in the
molecular mass of LTBP. Activation of TGF-
occurs by proteolytic
cleavage of LAP(29, 30) , while cleavage of LTBP
results in the release of latent TGF-
1 from the
matrix(13) . We investigated therefore whether proteinase
treatments resulted in processing of the LAP portion of the large
latent TGF-
s. Analysis of the samples by 15% SDS-PAGE under
non-reducing conditions indicated that degradation products of
1-LAP were detectable only in samples treated with the activating
proteinase plasmin (Fig. 6, middlepanel;
100 kDa).
Figure 6:
Activation of large latent TGF-1 by
proteinases: analysis by sodium deoxycholate-PAGE and immunoblotting.
Purified recombinant large latent TGF-
1 (10 µg/ml) was
incubated with human mast cell chymase (500 nM), leukocyte
elastase (50 nM), and cathepsin G (500 nM) at 37
°C for 4 h. Treatment with plasmin (1 µM) was included
as a positive control. Heparin (+) was included as a cofactor in
some reactions (10 µg/ml). The reactions were terminated by the
addition of proteinase inhibitors. The amount of 25-kDa active form of
TGF-
1 was assayed from the samples by 4-15% gradient sodium
deoxycholate-PAGE followed by immunoblotting with antibodies specific
for TGF-
1 (toppanel; see ``Materials and
Methods'' and caption to Fig. 1B for details).
Activation of TGF-
1 was also analyzed by Mv1Lu cell growth
inhibition and an ELISA that is specific for active TGF-
1 (R&
Systems), and is expressed as + or -. Migratory positions of
human recombinant large latent TGF-
1 (Latent) and active
TGF-
1 (Active) are indicated on the right. The
analyses of the respective samples by nonreducing 4-15% gradient
SDS-PAGE followed by immunoblotting with antibodies specific for
1-LAP (middlepanel) and LTBP (bottompanel) are also shown.
The present study was carried out to analyze the effects of
two important proteases, mast cell chymase and leukocyte elastase, on
matrix-associated latent TGF- complexes. Monolayer cultures of
human umbilical vein endothelial cells and amnion epithelial cells were
used as model system. Unlike fibroblastic cells, epithelial and
endothelial cells grow in culture as sheets of polygonal monolayer
cells and form a matrix resembling basement
membrane(57, 62) .
Similar levels of LTBP and
latent TGF-1 were produced by human umbilical vein endothelial
cells and fibroblasts. Sodium deoxycholate-PAGE immunoblotting
experiments indicated that the majority of the latent TGF-
produced by endothelial cells was in a large latent complex, containing
both LTBP and
1-LAP. Significant proportion of total LTBP and
large latent TGF-
1 produced were also detected in the
extracellular matrix of endothelial cells. Human amnion epithelial
cells produced significantly less LTBP and very little TGF-
1.
However, higher proportion of the produced LTBP was deposited to the
matrix. Small latent TGF-
1 was not detected in the matrix of
either cell type.
When a panel of proteinases and
glycosaminoglycan-degrading enzymes were tested for their ability to
release matrix-bound TGF-1, it was found that mast cell chymase
and leukocyte elastase effectively released LTBP and TGF-
1 from
the matrix of epithelial and endothelial cells with ED
(1
h, 37 °C) of 5 nM, as estimated from dose dependence
studies. The enzymes were approximately 10-fold more efficient than
plasmin (ED
50 nM). Concentrations of the
above enzymes in this order of magnitude are likely to exist in the
vicinity of cell surfaces following mast cell or neutrophil
degranulation or plasminogen
activation(63, 64, 65) . The kinetics of the
cleavage reactions, as well as the fact that LTBP fragments produced by
elastases, chymase, and plasmin differed in molecular mass, make it
likely that these proteinases act directly and not via the activation
of secondary proteinase(s). Significant levels of LTBP were not
released from the matrix by glycosaminoglycan-degrading enzymes
heparinase I and III and chondroitinase ABC, suggesting that LTBP is
not bound to glycosaminoglycan structures. The ineffectiveness of
bacterial collagenase to release LTBP suggests in turn that
matrix-associated latent TGF-
1 is not an integral part of collagen
fibers (for in vivo distribution, see (20) ). However,
our recent immunofluorescence studies reveal in fibroblasts a fibrillar
staining of LTBP that colocalizes with fibronectin and collagen type IV (66) .
The proteinases releasing LTBP and latent TGF-1
from the matrix display significant differences in substrate
specificity(67, 68, 69, 70, 71, 72) .
The ability of enzymes with divergent P1 residue specificities to
cleave LTBP and release it from the matrix supports the hypothesis (13) that there is a proteinase-sensitive hinge region in LTBP
between extracellular matrix and latent TGF-
binding domains. The
presence of these two functional domains in LTBP is also supported by
the fact that the ability of proteinases to release TGF-
1 from the
matrix correlated to the ability of the enzymes to cleave the
160-200-kDa fibroblast form of LTBP to a fragment whose size
resembled platelet LTBP (90-140 kDa).
To study the activation
of TGF-1, we developed a biochemical assay based on polyacrylamide
electrophoresis in the presence of sodium deoxycholate. TGF-
1
released from endothelial cell matrix by chymase and elastase was
biologically latent (>95%). Recombinant large latent form of
TGF-
1 was not activated by chymase or elastase. Plasmin (1
µM) activated approximately 1-2% of the large latent
complex, while the activation of TGF-
1 by cathepsin G was
marginal. Sodium deoxycholate-PAGE represents the first demonstration
of a specific biochemical assay for TGF-
activation, and can, in
addition, distinguish between small and large latent TGF-
complexes. Sodium deoxycholate-PAGE or analogous electrophoretic
methods will facilitate studies on different latent TGF-
complexes
in tissues and cultured cells. They could also be used in the study of
non-covalent protein-protein interactions in general.
Mast cells and
neutrophils are particularly rich sources of proteolytic enzymes in
peripheral tissues, and their granule proteinases participate in
connective tissue degradation in inflammation. Mast cells often
localize in the areas of uncontrolled tissue degradation, such as in
the shoulder region of human coronary atheromas(73) , in sites
of tumor invasion and metastasis(74, 75) , and in
rheumatoid joints(76, 77) . Mast cell proteinases
degrade the extracellular matrix directly(54, 72) ,
and via the activation of zymogens, such as procollagenase (55) and prostromelysin(78) . On the other hand, mast
cells have been suggested to have a role in a variety of fibrotic
conditions(79, 80) . LTBP and TGF- are present in
the endothelial basement
membrane(20, 60, 61) . During inflammatory
processes, latent TGF-
released from the subendothelial
extracellular matrix by mast cell chymase and leukocyte elastase could
specifically bind to and be activated at the surface of smooth muscle
cells, as suggested by Sato et al. ((33) ; see also (28, 29, 30, 31, 32, 33, 34, 35, 36) ).
Active TGF-
could contribute to the recruitment of macrophages,
resulting in inflammatory cell infiltration to the intima, and smooth
muscle cell proliferation, which are associated with the development of
atherosclerosis(81) . This hypothesis is strengthened by the
finding that the expression of TGF-
1 activity causes intimal and
medial hyperplasia in porcine arteries transfected with constitutively
active TGF-
1 gene(52) . The excessive activity of
proteinases and formation of feedback TGF-
1 could lead to
excessive remodeling (degradation and production) of extracellular
matrix in chronic inflammation, resulting in the degradation of normal
connective tissue and epithelia, and in the formation of granulation
tissue(4, 47, 48) .
Low concentrations
(10-100 nM) of plasmin release TGF-1 from the
matrix in a latent form(17, 60) , while high
concentrations (
1 µM) activate a fraction of latent
TGF-
1(29, 30) . Recent genetic evidence suggests,
however, that physiological mechanisms distinct from plasmin exist for
the release of TGF-
1 from the matrix and its activation. Mice
lacking TGF-
1 gene die shortly after weaning due to invasion of
inflammatory cells to multiple organs (46) , whereas loss of
plasminogen activator u-PA and t-PA function in mice does not severely
affect the immune system(82) , indicating that u-PA- and
t-PA-mediated plasminogen activation is not required for the expression
of TGF-
1 activity. Under physiological conditions plasmin is
susceptible to inhibition by multiple serum protease inhibitors, such
as
-antiplasmin and
-macroglobulin,
and the conversion of plasminogen to plasmin can be controlled by
plasminogen activator inhibitors. TGF-
induces plasminogen
activator inhibitor-1, which decreases the activation of
plasminogen(41) . In contrast, mast cell chymase and leukocyte
elastase are released from the respective cells as active,
heparin-bound enzymes that are relatively resistant to
inhibition(64, 83, 84, 85, 86) .
These properties make mast cell chymase and leukocyte elastase
attractive candidates for modulators of TGF-
bioactivity during
inflammatory reactions in vivo.
It is becoming increasingly
clear that a major step in growth factor signaling is the modulation of
growth factor and receptor activity by extracellular proteinases (see,
for example, (3) and (87) -92). In the case of
TGF-1, at least three specific proteolytic cleavages are likely to
regulate TGF-
1 activity in tissues, namely: 1) the release of
TGF-
1 from the matrix(13, 17) , 2) the activation
of TGF-
(29, 30, 31, 32, 33, 34) ,
and 3) negative regulation by the shedding of TGF-
-binding protein
betaglycan from the cell surface(92) . The present results
indicate that a number of serine proteinases can release TGF-
1
from the matrix of cultured human epithelial and endothelial cells,
resulting in the formation of a soluble pool of latent TGF-
1.
Soluble latent TGF-
1 could subsequently be activated at the
surface of cells expressing binding sites for latent
TGF-
1(33) . Regulation of TGF-
1 availability,
activity, and cellular response by proteinases and the modulation of
proteolytic activity by TGF-
1 (41, 42) support a
general model where proteinases and latent matrix-bound growth factors
are components of an extracellular signal transduction machinery that
directs tissue construction and remodeling and regulates the activity
of infiltrating immune cells. Disturbances in these control circuits
could operate in a variety of disease states affecting tissue
morphology and function, such as arteriosclerosis, cancer, fibrotic
diseases, and chronic inflammation.