(Received for publication, February 28, 1997, and in revised form, April 22, 1997)
From the Department of Pathology and Laboratory
Medicine, University of Wisconsin, Madison, Wisconsin 53706, § FibroGen Inc., South San Francisco, California 94080, and
the ¶ Maurice and Gabriela Goldschleger Eye Research Institute,
Tel Aviv University Faculty of Medicine, Sheba Medical Center,
Tel Hashomer 52621, Israel
Transforming growth factor-1 (TGF-
1)
induces increased extracellular matrix deposition. Bone morphogenetic
protein-1 (BMP-1) also plays key roles in regulating vertebrate matrix
deposition; it is the procollagen C-proteinase (PCP) that processes
procollagen types I-III, and it may also mediate biosynthetic
processing of lysyl oxidase and laminin 5. Here we show that BMP-1 is
itself up-regulated by TGF-
1 and that secreted BMP-1, induced by
TGF-
1, is either processed to an active form or remains as
unprocessed proenzyme, in a cell type-dependent manner. In
MG-63 osteosacrcoma cells, TGF-
1 elevated levels of BMP-1 mRNA
~7-fold and elevated levels of mRNA for mammalian tolloid (mTld),
an alternatively spliced product of the BMP1 gene, to a lesser extent.
Induction of RNA was dose- and time-dependent and
cycloheximide-inhibitable. Secreted BMP-1 and mTld, induced by TGF-
1
in MG-63 and other fibrogenic cell cultures, were predominantly in
forms in which proregions had been removed to yield activated enzyme.
TGF-
1 treatment also induced procollagen N-proteinase activity in
fibrogenic cultures, while expression of the procollagen C-proteinase
enhancer (PCPE), a glycoprotein that stimulates PCP activity, was
unaffected. In contrast to fibrogenic cells, keratinocytes lacked
detectable PCPE under any culture conditions and were induced by
TGF-
1 to secrete BMP-1 and mTld predominantly as unprocessed
proenzymes.
Bone morphogenetic protein-1 (BMP-1)1
copurifies from osteogenic bone extracts with transforming growth
factor- (TGF-
)-like proteins BMP-2 through -7 (1). Thus, it was
suggested that BMP-1, by structure an astacin-like protease, may
function in morphogenesis by activating TGF-
-like molecules (1).
Consistent with this possibility, BMP-1 has a domain structure similar
to, but shorter than, that of tolloid, a Drosophila protein
that appears to act in patterning of embryos by potentiating the
activity of decapentaplegic, a TGF-
family member (2, 3). The
mammalian BMP1 gene is now known to produce alternatively spliced
mRNAs for BMP-1 and for a longer protein, mammalian tolloid (mTld), which has a domain structure identical to that of Drosophila
tolloid (4).
Fibrillar collagen types I-III are synthesized as procollagens,
precursors containing N- and C-terminal propeptides that are cleaved
extracellularly to yield mature triple helical monomers capable of
associating into fibrils (for a review, see Ref. 5). Recently, BMP-1
was shown to be identical to procollagen C-proteinase (PCP) (6, 7), the
activity that cleaves the C-propeptides of procollagen types I-III
(8-10), and mTld has also been found to have PCP activity
(7).2 Demonstration of PCP activity,
however, does not preclude the possibility that BMP-1 and/or mTld may
also activate TGF--like proteins. In fact, it is becoming
increasingly apparent that products of the BMP1 gene play multiple
roles in matrix deposition. These include proteolytic activation by
fibrogenic cells of lysyl oxidase (11), an enzyme necessary to
formation of covalent cross-links in fibrillar collagens and elastin,
and biosynthetic processing by keratinocytes of laminin 5 (12), a major
basement membrane component of skin. PCP activity of BMP-1 is
stimulated ~10-fold by the procollagen C-proteinase enhancer (PCPE),
a glycoprotein that binds the type I procollagen C-propeptide (10).
However, possible involvement of PCPE in other biological activities of BMP-1 and mTld has not been examined.
TGF-1, prototype of the TGF-
superfamily, induces net increases
in the deposition of insoluble matrix by cells. This is accomplished by
effecting decreased production of proteases that degrade matrix and
increased production of (i) inhibitors for such proteases, (ii)
structural matrix components such as procollagen types I-III (13), and
(iii) lysyl oxidase (14). Expression of the genes for the three
polypeptide chains of laminin 5 is also up-regulated by TGF-
in
keratinocytes (15). The induction by TGF-
1 of gene products involved
in deposition of matrix in general, and of known and potential
substrates of BMP-1/mTld in particular, prompted us to examine whether
TGF-
1 also regulates BMP-1/mTld expression and/or the expression of
PCPE. Here we document the effects of TGF-
1 on BMP-1, mTld, and PCPE
expression in fibrogenic cells and keratinocytes and on levels of
cleavage of type I procollagen C- and N-propeptides. Mechanisms for the
post-translational regulation of BMP-1 and mTld activity are also
noted, and implications of the various data for the regulation of
matrix deposition are discussed.
MG-63 human osteosarcoma cells were purchased
from the American Type Culture Collection (Rockville, MD), MC3T3-E1
murine osteoblastic cells were obtained from Dr. Richard Wenstrup
(Children's Hospital Research Foundation, Cincinnati, OH), and human
AH1F neonatal foreskin fibroblasts and primary keratinocytes were
obtained from Dr. Lynn Allen-Hoffman (University of Wisconsin, Madison,
WI). MG-63, MC3T3-E1, and AH1F cells were maintained in Dulbecco's modified Eagle's medium with 10% heat-inactivated (30 min, 55 °C) fetal calf serum. MC3T3-E1 cells were supplemented with 1%
nonessential amino acids. For experiments in which RNA was prepared
from MG-63 cells, media contained 0.1% heat-inactivated fetal calf
serum. For experiments in which samples for immunoblots were prepared from MG-63, MC3T3, or AH1F cultures, media was serum-free unless otherwise indicated. Keratinocytes were maintained in 0.15 mM Ca2+ KGM BulletKit medium (Clonetics) with
30 µg/ml bovine pituitary extract. For experiments, pituitary extract
was omitted from media of TGF-1-treated and untreated control
keratinocyte cultures. For treatment with TGF-
1 (Austral
Biologicals), just-confluent cells were rinsed once with serum- or
pituitary extract-free media and then treated with vehicle (5 mM HCl) or TGF-
1. Unless otherwise noted, TGF-
1
treatment was 2 ng/ml for MG-63 cells and 10 ng/ml for other cell
types. Also unless otherwise noted, TGF-
1 treatments were 24 h
for cultures harvested for RNA and 48 h for cultures harvested for
proteins. Ascorbate treatment of cells was at 50 µg/ml.
A 471-bp probe for RNA sequences shared by
human BMP-1 and mTld has been described (4). The 1474-bp insert of
full-length human cDNA clone KT11 (16) was used as a probe for PCPE
RNA. Probe for human 1(I) collagen RNA was a 4.3-kb EcoRI
fragment from plasmid pHUC (17), and a 2.0-kb human
-actin probe was purchased (CLONTECH). Poly(A+) RNA was
prepared with the FastTrack kit (Invitrogen). 2 µg of poly(A+) RNA/lane was electrophoresed on 1.2% agarose, 2.2 M formaldehyde gels and transferred to Hybond-N+ membranes
(Amersham Corp.). Probes were radiolabeled to a specific activity of
4-6 × 109 cpm/µg by random priming (18) and
hybridized to blots in QuikHyb (Stratagene) at 65 °C for 1 h.
Blots were washed twice in 2 × SSC, 0.1% SDS for 10 min at room
temperature and then twice in 0.1 × SSC, 0.1% SDS for 20 min at
65 °C and exposed to Kodak X-Omat AR film with intensifying screens
at
70 °C. Autoradiograms were quantitated by scanning densitometry
(Biomed Instruments) of autoradiograms exposed for varying lengths of
time.
All antibodies were derived from polyclonal
rabbit antisera. Antisera LF-40, LF-41, and LF-67 for the human
pro-1(I) N- and C-propeptides and C-telopeptide, respectively, have
been described (19) and were kindly provided by Dr. Larry Fisher
(National Institutes of Health, Bethesda, MD).
To raise antibodies to full-length PCPE, sample enriched for 55-kDa PCPE by lysyl-Sepharose chromatography (10) was subjected to SDS-PAGE in unreduced 10% gels, and proteins were visualized with Coomassie Blue (20). The 55-kDa PCPE band was excised, equilibrated with SDS sample buffer (21) with 100 mM dithiothreitol, and electrophoresed on a 7% gel. After staining, gel pieces containing 55-kDa PCPE were equilibrated with PBS, crushed, and used for immunization as described (10). PCPE antibodies, isolated from an IgG fraction by adsorption to 55-kDa PCPE bound to nitrocellulose (22), were eluted with 5 mM glycine, 0.5 M NaCl, and 0.1% bovine serum albumin (pH 2.3) and neutralized with 1 M Na2HPO4.
Antibodies to the C termini of BMP-1 and mTld were raised against peptides CPHQLKFRVQKRNRTPQ and CLRYTSTKFQDTLHSRK, representing the final 16 residues of each respective protein plus an additional cysteine for coupling to keyhole limpet hemocyanin. Rabbits injected with the mTld peptide were boosted with the same peptide conjugated to ovalbumin to increase titers. BMP-1/mTld proregion antibodies, kindly provided by Dr. Mitch Brenner (FibroGen Inc.), were raised against peptide DLAEEDDSEPLNYKDPC, corresponding to residues 31-47 of the BMP-1 sequence (1), linked to keyhole limpet hemocyanin. Peptide antibodies were affinity-purified on columns of the appropriate peptide coupled to TC gel (Quality Controlled Biochemicals) via the cysteine thiol. They were eluted with 3 M MgCl2, 25% ethylene glycol and dialyzed against 10 mM sodium phosphate (pH 7.4), 20 mM NaCl.
Western BlotsProtease inhibitors (2.5 mM EDTA,
1 mM phenylmethylsulfonyl fluoride, 1 mM
p-aminobenzoic acid, 1 mM
N-ethylmaleimide) were added to harvested media, and
proteins were precipitated by adding trichloroacetic acid to 10%.
Pellets were washed with ice-cold acetone and then washed twice with
75% ethanol, 12.5 mM Tris (pH 7.5), dried, and resuspended
in SDS sample buffer with 5% -mercaptoethanol. Cell layers were
scraped into hot SDS sample buffer, as described (23). Media and cell
layer samples, equivalent to 5 × 105 fibrogenic cells
or keratinocytes per lane, were subjected to SDS-PAGE and transferred
to Immobilon-P membranes (Millipore) by electroblotting in 25 mM Tris, 192 mM glycine, 10% methanol at
4 °C. Blots were incubated ~14 h with primary antibody diluted 1:5000 in PBS, 1% bovine serum albumin, 0.05% Tween 20. After washing
three times with wash buffer (PBS, 0.05% Tween-20), blots were
incubated 1 h with horseradish peroxidase-conjugated donkey anti-rabbit IgG (Amersham) diluted 1:4000. Blots were then washed four
times with wash buffer, incubated with SuperSignal CL-HRP substrate
(Pierce), and exposed to film. Apparent molecular weights of bands were
estimated by comparison to electrophoretic mobilities of prestained
standards (Bio-Rad).
To quantitate relative amounts of BMP-1, gel lanes were loaded with
sample corresponding to the medium of 2.5 × 105
untreated MG-63 cells or to the medium of 1.25 × 104,
5.0 × 104, or 2.5 × 105
TGF-1-treated MG-63 cells. After SDS-PAGE, a blot was prepared, treated with antibody to the BMP-1 C terminus (as above), and then
incubated with 125I-labeled protein A (Amersham) for 1 h in PBS, 1% bovine serum albumin, 0.05% Tween-20, 1 mM
dithiothreitol. The blot was washed 8 h with wash buffer and
exposed to film, and the autoradiograph used as a template for excising
radioactive BMP-1 bands, which were counted in an A 800CD
counter
(Packard).
For digestion with peptide-N-glycosidase F, proteins were
precipitated from media with trichloroacetic acid (as above); denatured in 0.5% SDS, 1% -mercaptoethanol for 10 min at 100 °C; and
incubated 1 h at 37 °C with 500 units of
peptide-N-glycosidase F (New England Biolabs) in 50 mM sodium phosphate (pH 7.5), 0.1% SDS, 1% Nonidet P-40,
1 mM phenylmethylsulfonyl fluoride, 100 µg/ml soybean
trypsin inhibitor, 10 mM benzamidine; and the reaction was
stopped by adding 4 × SDS sample buffer and heating for 4 min at
100 °C.
14C-labeled procollagen substrate prepared
from chick embryo tendon cultures (8) was kindly provided by Dr. Darwin
Prockop (Allegheny University, Philadelphia, PA). Media tested for PCP activity contained 1 mM each phenylmethylsulfonyl fluoride,
p-aminobenzoic acid, and N-ethylmaleimide; was
concentrated 50-fold in Centriprep concentrators (Amicon); and then was
dialyzed against 50 mM Tris-HCl, 0.15 NaCl, 5 mM CaCl2. 5 µl of concentrated medium was
added to 0.125 µg of substrate in 5 µl of 0.1 M
Tris-HCl, 0.1 M NaCl, 5 mM CaCl2,
0.02% Brij 35 (pH 7.6) and incubated for 4 h at 35 °C. Reactions were stopped by adding 5 µl of 3 × SDS sample buffer and heating for 4 min at 100 °C prior to SDS-PAGE on nonreduced 6%
gels. Gels were fixed in 20% MeOH, placed in Amplify (Amersham) for 30 min, dried, and exposed to film. To quantitate PCP activity, percentage
of cleavage was calculated for each lane of an autofluorogram by
comparing signal remaining in the pro- band (procollagen in which
cleavage has not occurred in any of the three PCP cleavage sites) to
total signal (pro-
+ pro-
+ pro-
+ pN
1 +
1 + pN
2 +
2 + free C-propeptide bands) (see Fig. 5). The percentages of
cleavages achieved with media from cultures treated with TGF-
1, ascorbate, or TGF-
1 plus ascorbate were then compared with the percentages of cleavage achieved with media from untreated controls to
calculate -fold increases in PCP activity. Signals on autofluorograms were quantitated with an IS1000 digital video imaging system (Alpha Innotech).
RNase Protection Assays
To generate BMP-1/mTld-specific
riboprobe, a 1619-bp ApaI-EagI fragment was
excised from a baculovirus transfer vector containing full-length human
BMP-1 cDNA (6). The fragment, extending from nucleotide 615 of the
BMP-1 cDNA sequence (1) to an EagI site in the vector
polylinker, was inserted between pBluescript II KS+ (Stratagene)
ApaI and EagI sites and linearized at a
HincII site at nucleotide 1655 of the BMP-1 sequence (1).
This template produced a 617-base riboprobe of which 575 bases are
BMP-1-specific and 481 bases are specific for alternatively spliced
mTld RNA. For PCPE-specific riboprobe, a 329-bp fragment of PCPE
cDNA clone KT3 (16) was excised with EcoRI and
SacII and inserted between pBluescript II KS+
EcoRI and SacII sites. This template, linearized at the pBluescript XhoI site, produced a 391-base riboprobe,
329 bases of which correspond to nucleotides 557-885 of the human PCPE
cDNA sequence (16). Control riboprobe was generated with template
pTRI--actin-Human (Ambion). Uniformly 32P-labeled
riboprobes were generated by transcription with RNA polymerase T7,
hybridized to total RNA isolated with TRIzol (Life Technologies, Inc.),
and analyzed by digestion with RNases A and T1 and electrophoresis on
6% denaturing gels, as described (24).
To determine whether
levels of PCPE, BMP-1, and/or mTld mRNA are influenced by treatment
with TGF-, just-confluent MG-63 cultures were treated for 24 h
with 2 ng/ml TGF-
1. Levels of the ~3.0-kb BMP-1 mRNA and
~5.5- and ~4.1-kb mTld mRNAs (4) were all increased by TGF-
1
treatment (Fig. 1). Interestingly, mRNAs for the two
proteins did not increase to the same extent, with BMP-1 mRNA
increasing ~7-fold and both mTld mRNA forms increasing only
~4-fold. By comparison, levels of the 4.8- and 5.8-kb pro-
1(I) collagen mRNAs (25) increased ~9-fold, while those of the
-actin mRNA control did not change. In contrast to the induction
of BMP-1 and mTld mRNA, levels of PCPE mRNA were not increased
by TGF-
1.
Incubation of MG-63 cells with TGF-1 concentrations ranging from
0.01 to 10 ng/ml, showed induction of BMP-1 and mTld mRNAs to be
dose-dependent (Fig. 2A), with
substantial increases first noted at 0.5 ng/ml and maximum induction at
2 ng/ml. Induction of mTld and BMP-1 mRNAs was somewhat less at 10 ng/ml than at 2 ng/ml, possibly due to the toxicity of TGF-
1 at
higher concentrations (26). When kinetics of induction of BMP-1/mTld
mRNAs were examined by incubating MG-63 cultures in 2 ng/ml
TGF-
1 for varying times (Fig. 2B), substantial increases
first occurred at 12 h, with maximal levels attained at 24 h
post-treatment. Thus, induction was delayed compared with induction of
RNAs encoding a variety of extracellular matrix proteins, which
generally occurs within 3-5 h of TGF-
treatment of various cell
types (13). This delay suggested that induction of BMP-1/mTld mRNAs
may occur secondarily to an earlier event triggered by TGF-
1, a
possibility supported by experiments in which treatment of confluent
cultures with cycloheximide prior to TGF-
1-treatment showed protein
synthesis to be required prior to induction of BMP-1/mTld mRNAs
(Fig. 2C).
Levels of Secreted BMP-1 and mTld, but Not PCPE, are Up-regulated by TGF-
To determine whether
induction of steady state levels of BMP-1 and mTld mRNA in MG-63
cells was paralleled by increases in the cognate proteins, levels of
BMP-1 and mTld protein secreted into culture media were examined by
Western blots. Antibodies specific for the BMP-1 C terminus detected
bands of ~88 and ~77 kDa, both of which were strongly up-regulated
by TGF-1 (Fig. 3A). Antibody to the mTld C
terminus detected a ~130-kDa band that was up-regulated by TGF-
1
to a lesser extent than the anti-BMP-1-reactive bands. The relative
extent of induction of BMP-1- and mTld-specific bands, thus appeared
similar to the relative extent of induction of the cognate
mRNAs.
To quantitate the induction of secreted BMP-1, Western blots were
incubated with antibody to the BMP-1 C terminus and then with
125I-protein A, to allow subsequent excision and
quantitating of counts in BMP-1 bands. This method, which can provide
good quantitation of relative amounts of proteins (27), showed levels
of the 88-kDa BMP-1 band to be ~8-fold higher in TGF-1-treated
MG63 cultures than in untreated cultures (not shown). Thus, the level
of induction of secreted BMP-1 was similar to the level of induction of
cognate mRNA. The 77-kDa band was not detected on these blots (Fig.
3D, lane 3) and was determined to be unrelated to
BMP-1 (see below).
Ascorbate may stimulate production of type I procollagen at
transcriptional and posttranscriptional levels (28-30). Since we were
interested in mechanisms that might contribute to co-expression of
BMP-1 and type I procollagen, we examined whether levels of BMP-1 were
also up-regulated by ascorbate. Although the intensity of BMP-1 and
mTld protein bands did not appreciably increase in the presence of
ascorbate alone, intensities seemed somewhat higher in the presence of
TGF-1 plus ascorbate than in the presence of TGF-
1 alone (Fig.
3A). In contrast to BMP-1 and mTld, levels of secreted PCPE
did not increase in MG-63 cultures treated with TGF-
1, ascorbate, or
ascorbate plus TGF-
1 (Fig. 3A). Thus, as with BMP-1 and
mTld, levels of secreted PCPE paralleled levels of cognate
mRNA.
To ascertain whether results like those obtained with MG-63 cells were
common to other fibrogenic cells, analyses were done on MC3T3-E1
osteoblastic mouse cells and human dermal fibroblasts (Fig. 3,
B and C). Results obtained with these cells were
similar to those observed with MG-63 cells in that levels of secreted BMP-1 and mTld were elevated in the presence of TGF-1 and appeared to be slightly more elevated in the presence of TGF-
1 plus
ascorbate. As with MG-63 cells, levels of PCPE were not dramatically
changed in a consistent way by the addition of TGF-
1 and/or
ascorbate.
Some Western blots using BMP-1 C terminus antibody detected a ~77-kDa
band, in addition to the 88-kDa BMP-1 band, in MG-63 media (Fig. 3,
A and D, lane 2). This band, without
counterpart in MC3T3-E1 or fibroblast cultures, was not always detected
in MG-63 cultures treated with TGF-1 or TGF-
1 plus ascorbate and was absent upon culturing in the presence of 0.1% serum (Fig. 3D, lane 1) or when protein A was used instead of
secondary antibody (Fig. 3D, lane 3). The 77-kDa
MG-63 band was detected on blots when preimmune sera or mTld C terminus
antibody was used as primary antibody and by secondary antibody in the
absence of primary antibody (not shown). We therefore conclude that the
77-kDa MG-63 band is unrelated to BMP-1.
We next
examined whether induction of secreted BMP-1 and mTld in MG-63 cultures
was paralleled by induction of PCP activity, as evidenced by increased
cleavage of endogenous type I procollagen C-propeptides. Immunoblots
using antibody to the pro-1(I) N-propeptide (Fig.
4A, part a) did not detect
pN
1(I) chains in media of untreated cells or, surprisingly, in media
of cells treated with TGF-
1 alone, although the latter contained
relatively high levels of procollagen (Fig. 4A), BMP-1, and
mTld (Fig. 3A). In contrast, pN
1(I) chains were found in
media of ascorbate-treated cells, in a pro-
1(I):pN
1(I) ratio of
~1.0:0.3, and at higher levels in media of cells treated with
TGF-
1 plus ascorbate, with a pro-
1(I):pN
1(I) ratio of
~1.0:0.8 (Fig. 4A, part a).
Pro-1(I) C-telopeptide antibody, capable of detecting
1(I),
pN
1(I), and pC
1(I) chains (Fig. 4A, part
b), revealed relative levels of pro-
1(I) and pN
1(I) chains
in the various media, similar to those detected by the first antibody.
In addition, pC
1(I) and mature
1(I) chains were detected in media
of cells treated with TGF-
1 plus ascorbate. In the latter sample,
the ratio of unprocessed pro-
1(I) chains to chains from which the
C-propeptide had been cleaved (pN
1(I) +
1(I)) was ~1.0:2.7, a
value ~10-fold greater than the pro-
1(I):pN
1(I) ratio
(~1.0:0.3) detected in the ascorbate-treated sample by the same
antibody. Thus, although cultures treated with TGF-
1 plus ascorbate
contained substantially more substrate than cultures treated with
ascorbate alone, a greater fraction had undergone removal of
C-propeptides, indicating significantly increased PCP activity. A
diffuse band, about the size of pC
1(I), faintly detected by
C-telopeptide antibody in samples treated with TGF-
1 alone (Fig.
4A, part b) was not detected by
C-propeptide-specific antibody (see below) and probably represents
partially degraded pro-
1(I) chains.
The appearance of quantities of pC1(I) and
1(I) chains only in
media of MG-63 cells treated with TGF-
1 plus ascorbate, indicated
that procollagen N-proteinase (PNP) activity is also elevated under
these conditions. Highlighting this induction, pro-
1(I) C-propeptide
antibody detected pC
1(I) chains only in media of cells treated with
TGF-
1 plus ascorbate (Fig. 4A, part c). Since
the N-propeptides of procollagens I and III are cleaved by two
different PNP enzymes (31, 32), and since MG-63 cells produce
procollagen III (33), Western blots using antibody against the type III
procollagen C-propeptide were performed and showed that procollagen III
PNP activity is also elevated in the presence of TGF-
1 plus
ascorbate (not shown).
Since procollagen processing may help regulate incorporation of
monomers into fibrils (34), levels of processed 1(I) chains incorporated into MG-63 cell layers were also examined. Consistent with
results obtained from media, C-telopeptide antibody found high levels
of
1(I) chains in cell layers treated with TGF-
1 plus ascorbate,
while untreated cell layers or cell layers treated with TGF-
1 alone
did not contain detectable
1(I) (Fig. 4B, part b). Cell layers treated with ascorbate or with ascorbate plus TGF-
were enriched for
1(I) chains compared with media from the
same cultures (Fig. 4, A and B, parts
b). This enrichment may reflect preferential incorporation of
mature monomers into growing fibrils, rather than increased processing
in cell layers compared with media. Consistent with this likelihood,
pN
1(I), but not pC
1(I), chains were found in cell layers treated
with ascorbate or with TGF-
1 plus ascorbate (Fig. 4B,
parts a, b, and c), appearing to
confirm results previously obtained with in vitro
fibrillogenesis systems (34), suggesting that pN
1(I) but not
pC
1(I) chains are incorporated into growing fibrils. Unprocessed
pro-
1(I) in MG-63 cell layers (Fig. 4B) was likely intracellular, since procollagen is not thought to be incorporated into
fibrils (34). As in media, a diffuse band about the size of pC
1(I),
detected in some cell layers by C-telopeptide and N-propeptide
antibodies but not by C-propeptide antibody, probably represents
partially degraded pro-
1(I) chains.
It seemed
paradoxical that although secreted BMP-1 and mTld were elevated to only
slightly higher levels in MG-63 cultures treated with TGF-1 plus
ascorbate than in cultures treated with TGF-
1 alone (Fig.
3A), processing of endogenous procollagen was detectable
only in the former (Fig. 4). Ascorbate is a necessary cofactor for
enzymic hydroxylation of collagen prolyl residues, leading to a more
stable triple helix (35). Thus, to examine whether differences in
endogenous substrate might contribute to differences observed in levels
of processing, MG-63 media were assayed for PCP and PNP activity using
exogenous radiolabeled procollagen substrate. As with endogenous
substrate, PCP and PNP activities against exogenous substrate were
highest in media of cells treated with TGF-
1 plus ascorbate, as
evidenced by the generation of mature
-chains, processing
intermediates, and free C-propeptides (Fig. 5). However,
in contrast to results with endogenous substrate, PCP and PNP
activities against exogenous substrate were increased in media of cells
treated with TGF-
1 alone, to levels approaching those of cultures
treated with TGF-
1 plus ascorbate. Thus, in seven independent
experiments, one of which is shown in Fig. 5, the mean increase of PCP
activity against exogenous substrate was 2.4 ± 0.2-fold for
cultures treated with TGF-
1 plus ascorbate and 1.7 ± 0.1-fold
for cultures treated with TGF-
1 alone (values are mean ± S.E.). PCP activity in media of cultures treated with ascorbate alone
was virtually unchanged, 1.0 ± 0.1-fold, compared with untreated
controls. These results suggest that observed differences in processing
of endogenous substrate in cultures treated with TGF-
1 alone,
compared with cultures treated with TGF-
1 plus ascorbate, were at
least partly due to differences in the hydroxylation state and
conformation of the substrate.
Proteases of the
astacin family are synthesized as proenzymes with N-terminal proregions
that must be removed for activation (36, 37). Thus, the lesser
induction of PCP activity against exogenous substrate (~1.7-fold for
TGF-1-treated cultures), relative to levels of induction of secreted
BMP-1 detected by quantitative Western blots (~8-fold for
TGF-
1-treated cultures), might have been due to secretion of most
BMP-1 and mTld as inactive precursors. To ascertain whether this was
the case, antibody to proregion sequences common to BMP-1 and mTld
precursors was prepared and used to examine immunoblots of MG-63 medium
samples. This antibody did not recognize the major 88-kDa BMP-1 band
but did recognize a ~101-kDa band that also appears as a minor band
on immunoblots using the BMP-1 C terminus antibody (Figs.
6A and 3A). Thus, although a small
proportion is unprocessed, the majority of TGF-
1-induced BMP-1
secreted by the fibrogenic cells is in the processed active form. In
addition, the proregion antibody did not recognize the ~130-kDa band
detected by mTld C terminus antibody but did recognize a ~143-kDa
band (Fig. 6A). Thus, the majority of mTld secreted by
TGF-
-treated fibrogenic cells is also processed to the mature form,
while a small proportion, at levels undetectable with the mTld C
terminus antibody, is unprocessed. Interestingly, in repeated experiments of the type shown in Fig. 6A, media of MG-63
cells treated with TGF-
1 alone consistently had lesser amounts of
activated, and greater amounts of unprocessed, BMP-1 and mTld than did
cultures treated with TGF-
1 plus ascorbate. Thus, additional
processing of BMP-1 and mTld into mature forms seems to occur in the
presence of ascorbate, perhaps accounting for the higher levels of PCP activity against exogenous substrate in media of cultures treated with
TGF-
1 plus ascorbate than in media of cultures treated with TGF-
1
alone.
Previously, recombinant human BMP-1 produced in a baculovirus system by
this laboratory was shown to have high PCP activity and to be processed
to the mature form (6). Thus, BMP-1 secreted by TGF-1-treated
fibrogenic cells should have the same electrophoretic mobility as the
recombinant BMP-1, if in an active processed form. Electrophoretic
mobilities of BMP-1 from TGF-
1-treated fibroblast media and from the
baculovirus system were compared after treatment of samples with
peptide-N-glycosidase F to control for mobility differences
due to differences in Asn-linked glycosylation between insect and
mammalian cells (6, 38). Upon removal of Asn-linked carbohydrates, the
major form of BMP-1 secreted by TGF-
1-treated fibroblasts had the
same mobility as recombinant active BMP-1 (Fig. 6B),
bolstering the conclusion that most BMP-1 secreted by fibrogenic cells
in response to TGF-
1 is processed to the mature form.
It was of interest to
determine whether the patterns of expression and regulation of BMP-1
and mTld described above are also found in non-fibrogenic cells.
Keratinocytes were chosen for study, since they do not produce readily
detectable fibrillar collagens but do appear to utilize BMP-1 and/or
mTld for processing laminin 5 (12). Consistent with previous reports
that keratinocytes do not produce type I collagen (39), type I collagen
was not detected in media of keratinocytes treated or untreated with
TGF-1 and/or ascorbate by Western blots using pro-
1(I)
N-propeptide, C-propeptide, or C-telopeptide antibodies (not shown). In
addition, and in contrast to results obtained with fibrogenic cells,
PCPE expression by keratinocytes was undetectable by Western blot, regardless of culture conditions (Fig. 7A).
Also in contrast to results with fibrogenic cells, antibodies against
BMP-1 or mTld C termini did not clearly detect specific bands on
Western blots of medium samples from keratinocytes treated or untreated
with TGF-
1 and/or ascorbate (not shown). This indicated that if
keratinocytes secrete BMP-1 and/or mTld, then steady state levels in
media are significantly lower than in fibrogenic cell media.
We had noted in previous experiments that the proregion antibody was of
higher affinity than antibodies against the C termini of BMP-1 and
mTld. The proregion antibody was able to detect ~143- and ~101-kDa
bands, corresponding in size to unprocessed mTld and BMP-1 bands
observed in MG-63 cultures, in media of keratinocytes treated with
TGF-1 or with TGF-
1 plus ascorbate (Figs. 6A and 7B). The ~143-kDa band was barely detectable, and the
~101-kDa band was not detected, in media of untreated or
ascorbate-treated keratinocytes. Consistent with the observation that
keratinocytes secrete predominantly unprocessed mTld and BMP-1 in
response to TGF-
1, PCP activity against exogenous procollagen
substrate was at very low levels in media of keratinocytes treated or
untreated with TGF-
1 and/or ascorbate (not shown).
An RNase protection assay showed keratinocytes and MG-63 cells to contain comparable levels of mTld RNA but MG-63 cells to contain higher levels of BMP-1 RNA (Fig. 7C). Thus, secretion of predominantly mTld by keratinocytes and predominantly BMP-1 by MG-63 cells is reflected, at least in part, in levels of cognate RNA transcripts. RNase protection also demonstrated that absence of PCPE secretion by keratinocytes is reflected in a virtual absence of PCPE RNA (Fig. 7C).
The necessary action of BMP-1 and mTld in processing of matrix
components (6, 7, 12) and lysyl oxidase (11) implies that these
proteins play key roles in controlling the deposition of matrix in
developmental and homeostatic processes. Previously, however,
mechanisms for regulating functional expression of these key proteins
have not been explored. In this study we have demonstrated that
TGF-1 elevates levels of BMP-1 and mTld in fibrogenic cells and
keratinocytes. PNP activity against procollagens I and III was also
elevated by TGF-
1 in fibrogenic cells. Thus, in addition to
increasing the deposition of matrix through induction of matrix components, lysyl oxidase, and metalloprotease inhibitors, TGF-
1 also influences net matrix deposition by inducing the proteases that
process procollagens, laminin 5, and lysyl oxidase into mature forms.
The induction of BMP-1 and mTld by TGF-1, described here, is
particularly intriguing in the context of previous suggestions that
BMP-1- and tolloid-like proteins may activate TGF-
-like molecules
(1-3). TGF-
1 is itself secreted as a latent form (40), and the
possibility, therefore, exists of a positive feedback loop in which
BMP-1 and/or mTld activate, and are in return induced by, TGF-
1.
There is a precedent for such positive feedback loops in matrix
deposition, since the effects of TGF-
1 on matrix deposition are
amplified and prolonged through autoinduction by TGF-
1 of its own
expression (41). Preliminary studies with recombinant proteins suggest
that BMP-1 may indeed be capable of directly activating the TGF-
1
small latent complex.3
Removal of the proregion from astacin-like proenzymes is thought
necessary for the production of active forms of these enzymes (37).
Thus, persistence or removal of the proregion represents another
potential control point for regulating BMP-1 and mTld activities. In
the present study, the degree of processing of secreted BMP-1 and mTld
is shown to be cell type-specific, with predominantly processed forms
produced by TGF-1-treated fibrogenic cells and predominantly
unprocessed forms produced by TGF-
1-treated keratinocytes. Clearly,
the production of large amounts of activated BMP-1 and mTld would aid
fibrogenic cells in their highly specialized roles of producing large
quantities of fibrillar collagen matrix, especially in response to
TGF-
. It is less clear why keratinocytes produce predominantly
unprocessed, inactive forms of mTld and BMP-1 in response to TGF-
.
Consistent with this finding, however, is the observation that
processing of
2, the laminin 5 chain cleaved by mTld and/or BMP-1,
is delayed following secretion by cultured keratinocytes (12). Thus,
extracellular processing of mTld and BMP-1 may be a rate-limiting step
in the deposition of keratinocyte extracellular matrix, of which
laminin 5 is a major component (42, 43). In vivo, such
processing may be regulated by epithelial-mesenchymal interactions that
influence the production of matrix by basal keratinocytes (44).
Keratinocytes were found not to produce detectable amounts of PCPE,
suggesting that PCPE may not play a role in laminin 5 processing.
However, the possibility that, in vivo, PCPE may be provided
by dermal fibroblasts for this purpose has not been precluded.
Interestingly, the relative amounts of BMP-1 and mTld produced by cells were also found to be cell type-specific: fibrogenic cells produced relatively large amounts of BMP-1, while keratinocytes produced predominantly mTld, at both RNA and protein levels. The possible functional significance for the production of differing ratios of BMP-1 and mTld by different cell types remains to be determined, however, since a functional difference has yet to be discerned for these two protein products of the same gene.
Previously, the low levels of PCP activity detectable in tissues and in
cell culture systems have led to suggestions that the enzyme is either
secreted as an inactive precursor or co-expressed with an endogenous
inhibitor (8). Either of these possibilities might have explained the
discrepancy observed in the present study between the induction by
TGF-1 in MG-63 cultures of an ~8-fold increase in secreted BMP-1
and a ~2-fold increase in medium PCP activity against exogenous
substrate. However, since secreted BMP-1 and mTld were both found
predominantly as processed forms, this leaves the interesting
possibility of an endogenously produced inhibitor. Previously,
localized control over the activities of various proteases, including
degradative matrix metalloproteases such as stromelysins and
collagenases, has been shown to involve not only the processing of
proenzymes to mature forms but also the balance between levels of
activated enzyme and levels of specific inhibitors co-expressed by the
same cell types (45). Studies to determine the possible existence of
inhibitors for BMP-1, mTld, and related proteases (46) seem warranted
by results presented in the current study. Clearly, such inhibitors,
should they exist, could play roles in morphogenetic processes as
important as those of the proteases with which they interact.
A final point of interest relates to the observed processing of
endogenous procollagen in MG-63 cultures treated with TGF-1 plus
ascorbate and the absence of such processing in cultures treated with
TGF-
1 alone. Both cultures secreted high levels of similarly
processed BMP-1 and mTld. Moreover, the TGF-
1 plus ascorbate-treated
cultures had only slightly higher PCP activity against exogenous
substrate, corresponding to a slightly higher ratio of activated to
unprocessed BMP-1. One interpretation of these data is that fully
hydroxylated procollagen produced by ascorbate-treated cultures, or
supplied as exogenous substrate, is much better substrate for BMP-1
than is underhydroxylated procollagen produced in the absence of
ascorbate. Since underhydroxylated procollagen is not likely to be in a
compact triple helical form at 37 °C (35), these data might indicate
some conformational requirement for the cleavage of procollagen by PCP.
However, this would be in contrast to an earlier report (8) that found
PCP to cleave heat-denatured procollagen with about the same efficiency as native procollagen.