(Received for publication, August 15, 1995; and in revised form, October 18, 1995)
From the
The final enzymatic step required for collagen cross-linking is
the extracellular oxidative deamination of peptidyl-lysine and
-hydroxylysine residues by lysyl oxidase. A cross-linked collagenous
extracellular matrix is required for bone formation. The goals of this
study were to compare the transforming growth factor (TGF)-1
regulation of lysyl oxidase enzyme activity and steady state mRNA
levels to changes in COL1A1 mRNA levels in MC3T3-E1 osteoblastic cells.
TGF-
1 increased steady state lysyl oxidase and COL1A1 mRNA levels
in a dose- and time-dependent manner. The increase in lysyl oxidase
mRNA levels was transient, peaking at 12 h and 8.8 times controls in
cells treated with 400 pM TGF-
1. COL1A1 steady state mRNA
levels increased maximally to 3.5-fold of controls. Development of
increased lysyl oxidase enzyme activity was delayed and was of slightly
lower magnitude than the increase in its mRNA levels. This suggested
limiting post-translational processing of lysyl oxidase proenzyme.
Pulse-labeling/immunoprecipitation studies demonstrated slow proenzyme
secretion and proteolytic processing. Development and application of an
independent assay for lysyl oxidase proenzyme proteolytic processing
activity verified its proportionately lower stimulation by 400 pM TGF-
1. Thus, lysyl oxidase regulation by TGF-
1 in
osteoblastic cell cultures occurs at both pre- and post-translational
levels. This regulation is consistent with increased production of a
collagenous extracellular matrix.
Lysyl oxidase is the extracellular enzyme that catalyzes the
oxidative deamination of lysine and hydroxylysine residues in collagen
and elastin precursors to form
peptidyl--aminoadipic-
-semialdehyde, or
peptidyl-
-hydroxy-
-aminoadipic-
-semialdehyde,
respectively. Spontaneous condensation reactions of these resultant
aldehydes leads to the formation of lysine-derived cross-links found in
mature collagens and elastin(1) .
A cross-linked collagen
matrix is required for differentiation of osteoblastic cells and for
bone mineralization(2) . Type I collagen synthesis and
accumulation have been shown to be uncoupled processes in developing
MC3T3-E1 osteoblastic cells(3) . These cells accumulated
collagen in the extracellular matrix when the rate of collagen
synthesis was decreasing. This suggested post-translational control of
collagen fibril accumulation. Similarly, in primary rat calvaria
osteoblast cell cultures, TGF-1(
)-stimulated
extracellular collagen accumulation was controlled by unidentified
post-translational steps(4) . Lysyl oxidase-mediated control of
collagen accumulation in bone cell cultures has been
described(5) . In this study, treatment of chick osteoblast
cultures with the lysyl oxidase inhibitor,
-aminopropionitrile
(BAPN), reduced the accumulation of collagen in the cell layer by 50%.
Turnover of collagen deposited into the extracellular matrix was also
markedly increased in BAPN-treated cultures. Further evidence for the
importance of lysyl oxidase in bone formation was illustrated by in
vivo studies, where growing chicks and rats fed with BAPN
developed skeletal deformities known as
osteolathyrism(6, 7) .
TGF- increased
accumulation of extracellular matrix proteins in different cell and
organ culture models(4, 8, 9, 10) . In vivo, the highest levels of TGF-
were found in
platelets and bone(11, 12) . TGF-
-mediated
inhibition of osteoblastic cell growth was accompanied by increased
synthesis of type I collagen, fibronectin, and
proteoglycans(13, 14, 15) . TGF-
also
decreased the expression of extracellular matrix degrading enzymes, and
induced genes for protease inhibitors in different cell
types(16, 17) . Among the genes that were induced by
TGF-
in osteoblastic MC3T3-E1 cells, steady-state lysyl oxidase
mRNA levels increased almost 10-fold(18) .
Although lysyl
oxidase enzyme activity was not measured in the studies cited above,
taken together they raise the possibility that like collagens, lysyl
oxidase is a major target for TGF- in osteoblasts. If true, then
lysyl oxidase regulation by TGF-
could contribute to increased
collagen accumulation by osteoblastic cells stimulated by this growth
factor.
The biosynthesis of lysyl oxidase is complex and requires
numerous post-translational modifications, each of which could
potentially control activation of lysyl oxidase enzyme activity. Lysyl
oxidase is synthesized as a 50-kDa glycoprotein precursor, and is then
secreted and proteolytically processed to the mature 32-kDa
enzyme(19) . Recently, the cleavage site of the
NH-terminal propeptide region of the lysyl oxidase
precursor was reported(20) . Lysyl oxidase is a copper
metalloenzyme, and in addition contains an organic cofactor believed to
be a quinone derived from post-translational modification of a tyrosine
residue(1, 21) . The mechanisms of post-translational
constitution of the metal and organic cofactors with lysyl oxidase
proenzyme have not yet been defined.
In the present study, we report
that TGF-1 dramatically increased the levels of lysyl oxidase mRNA
and enzyme activity in MC3T3-E1 osteoblastic cultures. The effects on
steady-state lysyl oxidase and COL1A1 mRNA levels were compared.
Furthermore, we found that post-translational mechanisms may control
the expression of fully active lysyl oxidase.
The lysyl oxidase construct was cloned in three steps. Fragment I was lysyl oxidase coding region from bp 42 to bp 233 derived from a PstI fragment cloned into pUC 18 vector as described below (28, 29) . Fragment II was lysyl oxidase coding region from bp 234 to bp 939 (PstI/HindIII fragment). Fragment III was a HindIII fragment containing lysyl oxidase coding region from bp 940 through the end of the coding region, and extending to the first EcoRI site in the 3`-untranslated region. Fragments II and III were derived from the lysyl oxidase transcription vector pBSCOD (19) .
pUC 18 containing fragment I was digested with Eco0109I and recessed 3` termini were filled in using the Klenow fragment of E. coli DNA polymerase. Then the fragment was digested with PstI, yielding the 230-bp blunt/PstI fragment I. pBSCOD containing Fragments II and III was digested with PstI/HindIII, yielding Fragment II. This DNA was cloned into a PstI/HindIII-digested pMAL-p2 vector. pMAL-p2 DNA containing Fragment II was isolated from positive clones, digested with Xmn I/PstI, and ligated with blunt/PstI Fragment I, and correct clones were isolated. This pMAL-p2 plasmid containing Fragments I and II was digested with HindIII and 600-bp HindIII/HindIII Fragment III was cloned into this site. The resulting construct was lysyl oxidase cloned into pMAL-p2 (pLO/P2). As fusion protein expression from pLO/P2 was unacceptably low, we then recloned the NdeI/NcoI fragment of LO/P2 into the NdeI/NcoI site of pMAL-c2 resulting in construct pLO/C2. Limited DNA sequence analyses of these constructs revealed the correct nucleotide sequences at the junctions of lysyl oxidase Fragments I, II, and III. Detailed restriction mapping also confirmed the structure of these constructs.
pLO/C2 was used to
transform E. coli strain BL21 (DE3)pLys(S) (Novagen, Inc). Two
liters of logarithmic phase cells were induced with 0.3 mM
5-bromo-4-chloro-3-indoyl--D-glucopyranoside for 4.5 h,
collected by centrifugation at 5000
g, and stored at
-80 °C. The pellet was thawed and resuspended in 200 ml of 50
mM Tris, 2 mM EDTA, pH 8.0. At room temperature, 1.2
ml of 0.2 M phenylmethylsulfonyl fluoride, 25 ml of 10
mM MgCl
, and 10 mg of protease-free DNase I
dissolved in 8 ml of 0.15 M NaCl were added to the lysed cell
pellet and stirred for 10 min. The inclusion bodies were collected by
centrifugation, and then solubilized in 280 ml of 8 M urea
denaturing buffer, pH 7.8 (8 M urea, 20 mM sodium
phosphate, 500 mM sodium chloride, pH 7.8), and dialyzed
against 10 liters of 1 M urea denaturing buffer, pH 7.8, at 4
°C overnight, followed by several changes of amylose column buffer
(20 mM Tris-Cl, 200 mM NaCl, 1 mM EDTA, 10
mM
-mercaptoethanol) over 24 h. The soluble proteins were
then applied to an amylose column (New England Biolabs) as
described(31) . The fusion protein was eluted with column
buffer containing 10 mM maltose, with a typical yield of 6.4
mg from 2 liters of culture. The eluted fusion protein was further
purified by chromatography on hydroxylapatite as follows. Nine mg of
fusion protein dialyzed against 10 mM
KH
PO
, pH 7.2, was applied to a 10-ml
hydroxylapatite column (Sigma) equilibrated in the same buffer. The
column was successively washed with 40 ml of 10 mM
KH
PO
, pH 7.2, 40 ml of 500 mM
KH
PO
, pH 7.2. The column was then eluted with 6 M urea, 20 mM KH
PO
, pH 7.2.
The yield was 3.21 mg or 35%. The purified fusion protein was
exhaustively dialyzed into 50 mM Tris, pH 7.8, prior to use.
Purity was assessed by SDS-PAGE.
Figure 1:
Dose-dependent regulation of lysyl
oxidase and COL1A1 steady-state mRNA levels by TGF-1. MC3T3-E1
cells were cultured in the presence of TGF-
1 (0, 4, 40, and 400
pM). After 24 h, total RNA was extracted and subjected to
Northern blotting. Inset, autoradiograms of Northern blots
probed with
P radiolabeled lysyl oxidase and COL1A1 cDNAs,
and with DNA for 18 S ribosomal RNA (18S rRNA). Lane 1, 0
TGF-
1; lane 2, 4 pM TGF-
1; lane 3,
40 pM TGF-
1; lane 4, 400 pM TGF-
1. Black bars, lysyl oxidase; hatched bars, COL1A1.
Signals were quantitated by scanning laser densitometry and were
normalized to 18 S rRNA. Values represent mean ± S.D. obtained
from three scanning laser densitomentry determinations from one
representative experiment.
Figure 2:
Time-dependent effect of 400 pM TGF-1 on lysyl oxidase enzyme activity (A); and
steady-state levels of lysyl oxidase and COL1A1 mRNAs (B) from
parallel cultures. A, lysyl oxidase was measured in the
conditioned medium, using recombinant human
[
H]tropoelastin substrate. Values are mean
± S.E. of samples assayed in quadruplicate and normalized to
10
cells counted from the same plate of cultured cells. B, autoradiograms of Northern blots of total RNA (10
µg/lane) from MC3T3-E1 cells cultured in parallel in the absence (odd numbered lanes) or presence (even numbered
lanes) of 400 pM TGF-
1. Lanes 1 and 2, 3 h; lanes 3 and 4, 6 h; lanes 5 and 6, 12 h; lanes 7 and 8, 18 h; lanes 9 and 10, 21 h; lanes 11 and 12, 24 h. Data are from one of two replicate
experiments.
Figure 3:
Short-term pulse
labeling/immunoprecipitation/SDS-PAGE of lysyl oxidase proteins from
MC3T3-E1 cells pretreated with TGF-1 for 4 or 15 h. MC3T3-E1 cells
were pretreated without (control) or with 400 pM TGF-
1
for 4 or 15 h, and then pulse-labeled with
[
S]methionine for 3 h. Constant cpm of media
proteins (7
10
cpm) and cell layer extracts (2
10
cpm) were immunoprecipitated with anti-lysyl
oxidase antibody. Samples were then subjected to SDS-PAGE, and
autoradiography. Autoradiograms of immunoprecipitates from media (lanes 1-5): lane 1, control (4 h); lane 2, TGF-
1 (4 h); lane 3, control (15 h); lane 4, TGF-
1 (15 h); lane 5, as in lane 4 except
immunoprecipitation was performed in the presence of 50 µg of
unlabeled lysyl oxidase/maltose-binding fusion protein.
Immunoprecipitates from the cell layer (lanes 6-9): lane 6, control (4 h); lane 7, TGF-
1 (4 h); lane 8, control (15 h); lane 9, TGF-
1(15 h).
Molecular weight markers were Bio-Rad prestained low molecular weight
range markers.
Levels of
radioactive secreted proenzyme from growth factor pretreated cells were
slightly lower after 15 h compared to 4 h under these conditions of
pulse labeling (Fig. 3, lanes 2 and 4).
Control media had no detectable radiolabeled proenzyme (Fig. 3, lanes 1 and 3). No fully processed labeled 32-kDa
lysyl oxidase was detected under these conditions in control or growth
factor pretreated cultures (Fig. 3, lanes 1-4).
Consistent with previous studies(19) , the immunoprecipitations
were shown to be specific (Fig. 3, lane 5). These
findings suggest that secretion and extracellular proteolytic
processing of lysyl oxidase requires more than 3 h in MC3T3-E1 cells.
Thus, due to a large unlabeled intracellular lysyl oxidase pool after
15 h of TGF-1 pretreatment, secreted proenzyme was likely of lower
specific radioactivity than that secreted from cells pretreated for
only 4 h. This would account for the lower labeling of secreted
proenzyme at 15 h compared to 4 h of growth factor pretreatment.
As shown in Fig. 4(lanes 1-4), TGF-1 increased the pool
of lysyl oxidase precursor in the conditioned media in a time-dependent
manner. Similarly, a greater amount of labeled 32-kDa lysyl oxidase was
found at 18 h compared to 12 h. This is consistent with increased
enzyme activity observed after 18 h of growth factor treatment (Fig. 2). Densitometric scanning of autoradiograms from 12- and
18-h immunoprecipitates showed 3-fold increases in the 50 ± 5
kDa precursor (12 h, 689 ± 5.6; 18 h, 1, 964 ± 25.5) and
in the 32 ± 3 kDa form of lysyl oxidase (12 h, 378 ±
33.2; 18 h, 1, 121 ± 29.7) in TGF-
1-treated cells. The
accumulation of 50 ± 5-kDa lysyl oxidase only in the media of
TGF-
1-treated cultures suggests that the conversion to the 32
± 3-kDa species was limited by extracellular proteolytic
processing activity.
Figure 4:
Continuous
pulse-labeling/immunoprecipitation/SDS-PAGE of lysyl oxidase proteins
from MC3T3-E1 cells treated with TGF-1. MC3T3-E1 cells were
treated without (control) or with 400 pM TGF-
1 in the
presence of [
S]methionine continuously for 12
and 18 h. Constant cpm of media proteins (3
10
cpm)
and cell layer extracts (1
10
cpm) were
immunoprecipitated with anti-lysyl oxidase antibody. Samples were
subjected to SDS-PAGE. Autoradiograms of immunoprecipitates from the
media (lanes 1-4): lane 1, control (12 h); lane 2, TGF-
1 (12 h); lane 3, control (18 h); lane 4, TGF-
1 (18 h). Immunoprecipitates from the cell
layers (lanes 5-8): lane 5, control (12 h); lane 6, TGF-
1 (12 h); lane 7, control (18 h); lane 8, TGF-
1 (18 h).
Scanning laser densitometry of Fig. 4, lanes 1-4, revealed that the level of labeled 32
± 3-kDa lysyl oxidase increased by 13- and 20-fold in
TGF-1-treated cells compared to unstimulated cells at 12 and 18 h,
respectively. Assuming that the 32 ± 3-kDa molecular form of
lysyl oxidase was fully active and that the specific radioactivity of
methionine pools was comparable in these continuously labeled cultures,
corresponding increases in lysyl oxidase enzyme activity by TGF-
1
of 10-20-fold would be expected. Interestingly, we observed at
most only a 6-fold stimulation of enzyme activity at 18 or 24 h ( Table 1and Fig. 2). We conclude that lysyl oxidase is
unlikely to be fully active in TGF-
1-stimulated cultures (see
``Discussion'').
Results obtained from the cell layer suggest that relatively low levels of lysyl oxidase proenzyme accumulation occurred in this fraction compared to the media (Fig. 4). Moreover, absence of the 32 ± 3-kDa molecular form in the cell layer correlated well with the absence of enzyme activity. Thus, in these MC3T3-E1 osteoblastic cell cultures, lysyl oxidase was synthesized as a 50 ± 5-kDa precursor (Fig. 3), secreted and processed proteolytically to a 32 ± 3-kDa molecular species that remained soluble (Fig. 4). This is distinct from studies in neonatal rat aorta smooth muscle cells, and rat lung fibroblasts, where lysyl oxidase enzyme activity and the 32 ± 3-kDa molecular form were shown to accumulate in the cell layer after extracellular proteolytic processing(19, 32) .
Figure 5: Coomassie Blue-stained SDS-PAGE gel of lysyl oxidase/maltose binding fusion protein. Lane 1, Bio-Rad low molecular weight prestained markers; lane 2, 5 µg of partially purified fusion protein eluted from the amylose affinity column; lane 3, 1.5 µg of purified fusion protein eluted from hydroxylapatite.
Media obtained from MC3T3-E1 cells
treated for 24 h with 400 pM TGF-1 was incubated with
fusion protein in the presence and absence of 20 mM EDTA for
up to 3 h, and then analyzed by Western blotting. As shown (Fig. 6) MC3T3-E1 media converted the fusion protein to a
discrete 32 kDa ± 3-kDa molecular species clearly immunoreactive
with lysyl oxidase antibody in a time-dependent manner. This activity
was fully inhibited by 20 mM EDTA, suggesting that this
activity is a metalloenzyme (Fig. 6, lane 8).
Conversion to 32 kDa ± 3-kDa product was linear with respect to
time up to 50% conversion (Fig. 6). If reactions were carried
out beyond 50% conversion, production of 32-kDa product as a function
of time was no longer linear (Fig. 6, lanes 6 and 7 in inset were not plotted). Production of 32 ±
3-kDa product was also linear with respect to volume of culture medium
up to 50% conversion (not shown). Thus, linearity was maintained up to
50% conversion with respect to time of incubation, and volume of
MC3T3-E1 culture media. It is likely that loss of linearity beyond 50%
conversion was due to substrate depletion or product inhibition.
1,10-Phenanthroline (1 mM) inhibited production of 32 ±
3-kDa product by 87% (Fig. 6, lane 9). This further
supports that lysyl oxidase processing activity is a metalloenzyme(s).
Figure 6:
Linear time-dependent production of 32-kDa
immunoreactive lysyl oxidase from lysyl oxidase/maltose-binding fusion
protein substrate by conditioned MC3T3-E1 cell culture medium. MC3T3-E1
cells were treated with 400 pM TGF- for 24 h, and
200-µl aliquots of conditioned medium were incubated at 37 °C
with 13 µg of purified fusion protein in a final volume of 0.5 ml
of 50 mM Tris, pH 7.8. Reactions were stopped at intervals by
addition of 1 volume of SDS-PAGE sample buffer, and boiled for 3 min.
Fifty-microliter aliquots were subjected to Western blotting and
scanning laser densitometry. Inset, Western blot stained with
rabbit anti-bovine lysyl oxidase and alkaline phosphatase-coupled goat
anti-rabbit IgG visualized with nitro blue
tetrazolium/5-borom-4-chloro-3-indoyly phosphate Western Blue substrate
(Promega)(28, 40) . Lane 1, zero time; lane 2, 20 min; lane 3, 40 min; lane 4, 60
min; lane 5, 90 min; lane 6, 120 min; lane
7, 180 min; lane 8, same as lane 7 except
incubations also contained 20 mM EDTA; lane 9, same
as lane 7 except that incubations contained 1 mM 1,10-phenanthroline. The diffuse 65-kDa band is a nonspecific
artifact previously noted(40) . Replicate scans did not vary by
more than 5% conversion. Markers were Bio-Rad prestained low molecular
weight standards.
NH-terminal analysis was performed on the 32 ±
3-kDa product generated by culture media from TGF-
1-stimulated
cells (see ``Methods and Materials'' for details). As shown
in Table 2, the analysis is consistent with the sequence
Asp-Asp-Pro-Tyr-Ser. This is the same NH
-terminal sequence
identified for active lysyl oxidase(20) . This sequence does
not occur in the maltose-binding protein. Thus, the Western blot assay
detects activities that result in cleavage of the lysyl oxidase fusion
protein at the correct site.
Processing protease activity as a
function of time of TGF-1 treatment of MC3T3-E1 cells was
determined under valid assay conditions established above. These
results were compared to changes in lysyl oxidase enzyme activity in
the same media. Lysyl oxidase enzyme activity and processing protease
activity were not changed by 400 pM TGF-
1 after 3-6
h of treatment (Fig. 7). In contrast, from 12 h, both activities
were higher in media from growth factor-treated cultures.
Interestingly, the maximum increase in lysyl oxidase enzyme activity
was greater than the processing protease. Thus, at 15 h, lysyl oxidase
activity was 3.4-fold of control levels; whereas processing activity
was increased 1.7-2-fold. These results demonstrate that
increased lysyl oxidase enzyme activity induced by TGF-
1 was
accompanied by increased proteolytic processing activity. The degree of
stimulation of proteolytic processing activity by TGF-
1 was less
than the degree of stimulation of lysyl oxidase enzyme activity. These
results support the concept that proteolytic processing of lysyl
oxidase may partially limit the production of fully active 32 ±
3-kDa lysyl oxidase in these osteoblastic cell cultures.
Figure 7:
Time study of lysyl oxidase processing
activity (proteinase) and lysyl oxidase enzyme activity in MC3T3-E1
cells treated with no TGF-1 (closed circles) or with 400
pM TGF
1 (open boxes). MC3T3-E1 cells were
treated with 0 or 400 pM TGF-
1 as described under
``Methods and Materials.'' Plates were harvested at
intervals, and proteinase (see Fig. 6) and lysyl oxidase enzyme
activities were determined from the conditioned media, normalized to
cell number determined from the same plate. Values for lysyl oxidase
activity are means ± S.E. of quadruplicate assays. Values for
processing proteinase activity are means of duplicate determinations
± S.E. The volume of media assayed for processing proteinase
activity was 125 µl. This enabled all assays to be in the linear
range.
TGF-1 is present in high amounts in bone(12) ,
and has been shown to stimulate the synthesis of type I collagen,
fibronectin, and proteoglycans by osteoblastic
cells(14, 15) . Type I collagen synthesis and
accumulation have been shown to be uncoupled processes in developing
MC3T3-E1 cells(3) . Moreover, TGF-
1-stimulated collagen
accumulation in cell layers of rat calvaria osteoblastic cultures was
found to be partially controlled by unidentified post-translational
mechanisms(4) . In a separate study, lysyl oxidase mRNA was
found to increase 8-fold in MC3T3-E1 cells treated with
TGF-
1(18) . Recently, lysyl oxidase was implicated in
regulating collagen accumulation, and in decreasing collagen turnover
in mineralizing chick osteoblastic cultures(5) . It was
suggested that collagen cross-linking increases irreversible retention
of collagen fibrils in the extracellular matrix. This would amplify the
efficiency of collagen insolubilization, reduce the pool of soluble
collagen, and thereby decrease collagen susceptibility to proteolytic
degradation.
In the present study, we investigated whether lysyl
oxidase, like COL1A1 is a major target for TGF-1 in MC3T3-E1
osteoblastic cultures. We now report that TGF-
1 significantly
increased steady state lysyl oxidase mRNA levels, lysyl oxidase
proenzyme, and its extracellular proteolytic processing, in a dose- and
time-dependent manner. Increases in lysyl oxidase mRNA by 400 pM TGF-
1 were followed to a lesser degree by increases in enzyme
activity. Moreover, we found a 6-h delay between the growth
factor-stimulated peak in lysyl oxidase steady state mRNA levels and
enzyme activity. This suggests that lysyl oxidase biosynthesis is slow,
and may be limited by post-translational steps in MC3T3-E1 osteoblastic
cells.
Post-translational control of lysyl oxidase was supported by
short-term pulse-labeling/immunoprecipitation experiments following
TGF-1 treatment. Due to the short-term pulse labeling and low
level of processing protease(s), the resulting media (extracellular)
labeled lysyl oxidase were not processed to the 32-kDa molecular
species. Cell-associated radiolabeled lysyl oxidase proenzyme was
increased in proportion to TGF-
-dependent changes in steady state
lysyl oxidase mRNA levels, indicating that there was no block of
translation of lysyl oxidase pro-protein in TGF-
1-stimulated
cells. Experiments with long periods of continuous pulse labeling
resulted in increased accumulation of both 50-kDa precursor and 32-kDa
mature enzyme in the media. This supported the concept that lysyl
oxidase processing protease(s) partially limited production of the
32-kDa molecular form of lysyl oxidase.
We produced the lysyl oxidase proenzyme as a fusion protein with the maltose-binding protein in E. coli in order to develop an independent assay for lysyl oxidase processing protease(s). Interestingly, the maximum increase in lysyl oxidase enzyme activity (3.4-fold of control) at 15 h was greater than the processing protease activity (1.7-2-fold of control). This finding supported the pulse-labeling studies suggesting that proteolytic processing of lysyl oxidase proenzyme may partially limit the production of the 32-kDa molecular form of lysyl oxidase. Moreover, the inhibition by EDTA and 1,10-phenanthroline, confirmed the notion that this activity is a metalloproteinase(19, 20) .
400 pM TGF-1 caused an increase in steady state lysyl
oxidase mRNA and in both the 32 kDa ± 3-kDa and 50 ±
5-kDa kDa lysyl oxidase protein molecules, whereas the increase in
enzyme activity occurred to a lower extent. These findings may be
related to previous studies where a catalytically inactive 32-kDa form
of lysyl oxidase was isolated from bovine aorta(33) , and to
observations indicating that purified lysyl oxidase is not fully active (34) . Furthermore, TGF-
1 treatment might not have
increased the levels of proteins involved in copper transport and
metabolism, which could also have impaired development of full lysyl
oxidase enzyme activity(35, 36, 37) . It is
also currently unknown whether the 50-kDa molecule secreted by cultured
cells is catalytically active, although indirect evidence suggests that
this molecular form is inactive. Thus, cell cultures exhibiting lysyl
oxidase activity in the cell layer such as neonatal rat aorta smooth
muscle cells and rat lung fibroblasts have been shown to accumulate an
insoluble 32-kDa form of lysyl oxidase(19, 32) . In
contrast, cultures such as the MC3T3-E1 cells grown as described in the
present study accumulate no 32-kDa lysyl oxidase in the cell layer, and
exhibit no detectable activity in this fraction. All activity was found
in the medium. Taken together these studies show that lysyl oxidase
activity correlates well with the presence of the 32-kDa molecular form
of lysyl oxidase, and not with the 50-kDa molecular form. It is of
interest, however, that an active 45-kDa lysyl oxidase has been
demonstrated in rat skin tissues(38) .
As MC3T3-E1 cells produce relatively high levels of soluble lysyl oxidase enzyme activity, and 32 kDa- and 50 kDa-immunoreactive proteins, they may provide a source of lysyl oxidase that will allow direct analysis of the activity of these different molecular forms. Elucidation of the mechanisms of activation of lysyl oxidase activity in osteoblasts is required in order to evaluate the ultimate importance of these pathways in bone formation and pathology. It is of interest that lysyl oxidase dependent cross-linking has been shown to be decreased in human osteoporotic bone, and is likely related to the known bone weakness associated with this disease(41) .
MC3T3-E1 cells have been
shown to undergo phenotypic changes characteristic of the three
proposed stages of osteoblast differentiation, resulting in the
production of a mineralized extracellular
matrix(3, 39) . We have now shown that TGF-1
up-regulated lysyl oxidase activity and mRNA levels consistent with
COL1A1 mRNA regulation in preconfluent MC3T3 E1 cells. It will be of
interest to further these studies in long-term mineralizing organ and
cell cultures, analyzing for phenotype- and growth factor-dependent
changes in lysyl oxidase regulation and collagen cross-linking and
accumulation. Such studies may reveal minimum and optimum levels of
lysyl oxidase activity required for collagenous matrix accumulation.
These experiments would further define possible connections between the
regulation of lysyl oxidase and TGF-
1-mediated increases in bone
extracellular matrix.