(Received for publication, December 29, 1995)
From the
To examine the autocrine effects that an organizing
extracellular matrix has on osteoblast precursors, we created MC3T3-E1
cell lines that stably expressed pro-1(I) collagen chains with a
truncated triple helical domain. Cells that had incorporated the
pro-
1(I) expression plasmid (pMG155) expressed shortened
pro-
1(I) transcripts at high levels and efficiently secreted the
expression gene products into culture media. Those cells lost over 30%
of newly deposited collagenous matrix compared with virtually no loss
in control cultures, and media from the abnormal cells had qualitative
differences in matrix metalloprotinase production. Electron micrographs
strongly suggested that type I collagen molecules containing the
truncated pro-
1(I) chains dramatically interfered with collagen
fibrillogenesis in newly forming osteoblast matrix. Abnormal collagen
fibrillogenesis was also associated with altered characteristics of
cellular differentiation in that abnormal cells displayed a delayed and
attenuated increase in alkaline phosphatase activity. Surprisingly,
synthesis of osteocalcin was more than 5-fold higher than control
cultures. These findings demonstrate that osteoblasts require a
normally structured collagenous matrix for up-regulation of alkaline
phosphatase activity. However, in the presence of rapid turnover of
osteoblast matrix, osteocalcin gene expression may be up-regulated in
response to local signals by an unknown mechanism.
Since techniques have become available for isolation and analysis of osteogenic precursor cells in culture(1, 2, 3, 4, 5, 6) there has been considerable interest in using these cell culture systems to understand the controlling events that regulate cessation of cell division and the initiation of cellular events that are the hallmarks of differentiation. In both primary osteoblasts (2, 3, 5, 7) and in the cloned mouse calvarial cell line MC3T3-E1(1, 4, 6) , a period of rapid cell division is followed by a transitional period that is characterized by deposition of an insoluble, type I collagen-rich extracellular matrix and an initial rise in alkaline phosphatase activity. Later, a third stage occurs, beginning approximately 2 weeks after plating, which is characterized by further increases in ALP activity, expression of osteocalcin, and mineral deposition.
There is both in vitro and in vivo evidence that deposition of a collagenous matrix in the transitional stage may regulate or at least have a permissive effect on the third stage of maturation of the osteoblast phenotype. Cultured osteoblast precursor cells grown in the absence of ascorbate, which is necessary for collagen deposition, fail to differentiate(2, 4, 6, 8, 9) ; a similar ascorbate-dependent effect has been observed in cultured chondrocytes(10, 11) . MC3T3-E1 cells grown in the presence of the collagen synthesis inhibitor 3,4-dehydroproline also fail to show bone-specific gene expression(4, 9, 12) . In addition, the hypothesis that type I collagen-cell interactions play a role in osteoblast maturation is supported by the analysis of bone tissue from individuals with the heritable disorder osteogenesis imperfecta whose cells secrete type I collagen that is either defective in structure and/or decreased in amount. Histological and biochemical studies of osteogenesis imperfecta bone have detected several abnormalities, including increased osteoblast number(13, 14) , decreased staining for alkaline phosphatase activity(15) , and decreased deposition of noncollagenous extracellular matrix proteins in bone(16, 17) .
Collectively, the above findings
suggest that production of a collagen-rich extracellular matrix by bone
cells is required for limitation of cell growth by some mechanism of
feedback inhibition and for expression of alkaline phosphatase and
possibly other osteoblast products that are associated with
differentiated cells. Because it is not certain that ascorbate
deprivation or proline analogs were completely specific in their effect
on collagen matrix formation, we examined the osteoblast phenotype in
clones of MC3T3-E1 cells that synthesize shortened pro-1(I) chains
encoded by a human pro-
1(I) minigene(18) . The shortened
pro-
1(I) chains contain a normal carboxyl propeptide, a truncated
triple helical domain, and most of the amino-terminal propeptide. Mice
transgenic for this minigene have a brittle bone phenotype resembling
human osteogenesis imperfecta(19, 20) . Type I
collagen molecules containing this shortened pro-
1(I) chain are
secreted into the extracellular matrix resulting in increased turnover
of collagenous matrix and virtual destruction of the normal collagen
fibrillar architecture. Cells expressing the minigene also have
decreased alkaline phosphatase activity but significantly elevated
expression of osteocalcin. These studies use a dominant negative
collagen mutation to disrupt collagen fibrillogenesis in a more
targeted way than ascorbate deprivation or chemical inhibition of
collagen synthesis and provide evidence that normal collagen fibrillar
architecture is a necessary prerequisite for transition of MC3T3-E1
cells from immature dividing cells to a more mature state.
We tested the ability of MC3T3-E1 cells to stably express a
human pro-1(I) minigene construct (pMG155) that contained genomic
sequences including 2.5 kilobases of the pro-
1(I) promoter and
exons 1-5 and 47-52(18) . Cells were transfected
with pMG155 and the neo-containing expression plasmid pCDNA3.
G418-resistant clones were analyzed by Northern blot analysis for
expression of shortened pro-
1(I) transcripts (Fig. 1A), which were found in approximately one-half
of G418-resistant clones. Immunoblot analysis of procollagens harvested
from media and cell layer indicated that contrary to expectation, type
I collagen molecules incorporating the minigene product are efficiently
secreted from MC3T3-E1 cells rather than being degraded intracellularly (Fig. 1B). When several minigene expressing clones were
analyzed, there was surprisingly little heterogeneity in the expression
of the minigene product at the level of steady-state RNA or secreted
protein; expression either approached that of the endogenous
pro-
1(I) gene product or was undetectable even on prolonged
exposures of Northern blots.
Figure 1:
A, hybridization of a human
pro-1(I) collagen cDNA to 10 µg of total cellular RNA from
untransfected MC3T3-E1 cells (lane 1) and cells from clone C3 (lane 2) and clone F6 (lane 3), which were
transfected with pMG155 and pCDNA3 and selected with G418.
G418-resistant clones either had easily detectable levels of expression
of the minigene encoded in pMG155 (arrow) as in clone C3 or
else had only the endogenous pro-
1(I) bands (brackets). B, immunoblot of samples from media (lanes 1-3)
and cell layer (lanes 4-6) from untransfected MC3T3-E1
cells (lanes 1 and 4), clone C3 (lanes 2 and 5), or clone F6 (lanes 3 and 6). After
electrophoretic separation, proteins were visualized with LF41, an
antipeptide antiserum raised against the pro-
1(I) collagen
C-propeptide that recognizes human and murine procollagen chains. In
clone C3 but not clone F6 there is a low molecular weight band (arrow) detected with LF41. The low molecular weight band
representing the shortened pro-
1(I) product of pMG155 was
efficiently secreted into culture medium of clone C3 (lane
2).
When culture medium is supplemented
with 50 µg/ml ascorbate and 5 mM -glycerol phosphate,
MC3T3-E1 cells deposit a type I collagen-rich extracellular matrix that
increases in a linear manner and is first measurable at the end of the
first week of culture. Compared with cells from a representative
control clone F6 (that had been simultaneously co-transfected with
pCDNA3 and pMG155, selected with G418, and subsequently found not to
express the minigene), cells from the minigene expressing clone C3
deposited approximately the same amount of collagen in culture dishes
as measured by total hydroxyproline content (Fig. 2). To
determine whether secretion of abnormal type I collagen molecules
reduced the stability of collagen fibrils formed in the extracellular
matrix, we performed pulse-chase studies in which cells were labeled
for 48 h with [
H]proline beginning at 12 days
after plating. Rates of disappearance of
[
H]hydroxyproline from the insoluble cell layer
were measured for a 2-week chase period. Cultures expressing the
pro-
1(I) minigene retained only 66.3 ± 4.7% of newly
deposited [
H]hydroxyproline in osteoblast matrix
during the 2-week chase compared with 97 ± 7.5% in control
cultures (Fig. 3). Electron microscopy was performed to
determine whether the relative instability of collagenous matrix
produced by pro-
1(I) minigene-expressing cells could be related to
abnormal collagen fibrillar structure. Images of collagen fibrils
synthesized by control cells clearly show collagen fibrils recognizable
by 67-nm periodicity that results from the nonintegral quarter stagger
array of type I collagen monomers in fibrils; cells expressing the
pro-
1(I) minigene synthesize collagen fibrils that were thinner
and more disorganized and completely lacked the 67-nm pattern seen in
controls (Fig. 4) In an attempt to determine whether the loss of
[
H]hydroxyproline from the cell layer could be
explained by increased rates of collagen degradation, we examined the
production of gelatinases in culture media from minigene-expressing
cells by gelatin-substrate zymography. Compared with media from control
F6 cells, media from two different minigene-expressing clones (C3 and
C2) had higher rates of gelatinase activity overall and demonstrated
much higher expression of an as yet uncharacterized 38-kDa gelatinase (Fig. 5). In spite of the higher turnover of type I collagen in
the matrix, there was no evidence of compensatory up-regulation of the
endogenous pro-
1(I) collagen gene (Fig. 1, A and B). Secretion of osteonectin, decorin, and bone sialoprotein,
three other proteins that are deposited into osteoblast matrix and are
thought to be bound to or adjacent to collagen fibrils (28, 29, 30, 31, 32) , was
likewise not increased over control (Fig. 6).
Figure 2: Determination of hydroxyproline content of the cell layers of C3 cells (black, expressing the procollagen minigene) and control F6 cells (hatched). Cell layers were harvested, and proteins were hydrolyzed with 6 N HCl prior to spectrophometric analysis(22) . Error bars reflect standard error of the mean, n = 3.
Figure 3:
Percentage of
[H]hydroxyproline retained in the cell layer of
C3 cells (black, expressing the pro-
1(I) collagen
minigene) and F6 control cells (hatched) after labeling with
[
H]proline for 48 h at days 12-14 after
passage followed by chase with unlabeled media. Retention value was
arbitrarily set at 100% at day 0 of chase. Error bars reflect
standard error of the mean, n =
5.
Figure 4:
Electron
micrographs, at 60,000, of the cell layer of control cells
(clone F6, left) or cells that expressed the pro-
1(I)
minigene (clone C3, right). The 67-nm periodicity apparent in
the control collagen fibrils is absent in fibrils synthesized by
abnormal cells.
Figure 5: Gelatinases produced by mutant and control MC3T3-E1 clones. Conditioned media from mutant osteoblast clones (C3, lanes 1 and 2; and C2, lanes 3 and 4) or a control clone (F6, lanes 5 and 6) were analyzed by gelatin-substrate zymography. The 38-kDa gelatinase produced at high levels by mutant osteoblasts is denoted by the arrow.
Figure 6:
Immunoblots of samples from media of clone
C3 and clone F6 in which immobilized proteins harvested at days 8 and
21 after passage were reacted with LF113, antiserum to murine decorin
core protein (arrow), before and after digestion of
saccharides with chondroitinase (top), with LF 23, antiserum
to murine osteonectin (middle), or with LF87, antiserum to rat
bone sialoprotein (bottom). There were no significant
differences in band density between the control F6 and the pro-(I)
collagen minigene expressing C3 cells at either time
point.
We then
examined alkaline phosphatase activity in control and
minigene-expressing cells to determine whether abnormal collagen
fibrillogenesis had an effect on osteoblast differentiation. In the
presence of ascorbate and 5 mM -glycerol phosphate,
MC3T3-E1 cells in culture show a time-dependent decrease in cell growth
and a subsequent increase in alkaline phosphatase activity beginning
about 9 or 10 days after plating(4, 6) . There was
significant clonal variation in both basal and maximal levels of
alkaline phosphatase activity found in all clones, but the level of
maximal activity (attained between 21 and 28 days after plating)
correlated with basal activity, which was constant between days 2 and 8 (Fig. 7). Four control clones had maximal alkaline phosphatase
activity that was a mean 6.9 ± 1.5-fold increase over basal
activity, compared with a mean -fold increase of 2.2 ± 0.47 in
four minigene-expressing clones.
Figure 7: Basal and maximal levels of alkaline phosphatase activity, expressed in µmol of p-nitrophenol produced per min per cell, of four minigene-expressing MC3T3-E1 cell clones (open symbols) and four control clones (closed symbols). Mean -fold increase in control clones was 6.9 ± 1.5 over basal activity, compared with 2.2 ± 0.47 in four minigene-expressing clones.
To determine whether the abnormal collagenous matrix also attenuated expression of osteocalcin, another commonly used marker of osteoblast differentiation, we then measured secretion of osteocalcin by radioimmunoassay using antisera to mouse osteocalcin. Surprisingly, cells from the minigene-expressing clone C3 had five to six times more osteocalcin than control in culture media at days 14, 21, and 28 after plating (Fig. 8A). Because we were concerned that the elevated levels of osteocalcin might merely reflect osteocalcin released into culture media from the high turnover matrix rather than newly synthesized protein, we compared steady-state osteocalcin mRNA levels as well; differences in mRNA levels between control and abnormal cells either reflected or exceeded differences found in secreted osteocalcin measured by radioimmunoassay (Fig. 8B). To determine whether the increase in osteocalcin synthesis and steady-state RNA resulted from the abnormal matrix produced by the presence of truncated collagen chains in the matrix or was merely due to clonal variation in osteocalcin expression in MC3T3-E1 cells, steady-state osteocalcin mRNAs were examined by scanning densitometry of Northern blots in a total of four minigene-expressing and four control MC3T3-E1 clones. While there was some clone-to-clone variation in osteocalcin gene expression, clear differences in the pattern of osteocalcin expression were apparent (Fig. 9); much more osteocalcin mRNA was present in minigene-expressing clones, particularly at days 14 and 21 after passage. By day 28, consistent differences in steady-state levels of osteocalcin mRNA between minigene-expressing and nonexpressing clones were less apparent (data not shown).
Figure 8:
A,
osteocalcin concentration measured by radioimmunoassay at 14, 21, and
28 days after passage in culture media of C3 cells (black,
expressing the pro-1(I) collagen minigene) and F6 control cells (hatched). Error bars reflect standard error of the
mean, n = 3. B, hybridization of a murine
osteocalcin cDNA to 10 µg of total cellular RNA harvested from
clone C3 (M), and the control clone F6 (C) at days 3,
7, 11, 14, and 28 after plating. The approximately 0.6-kilobase
osteocalcin message (arrow) was detected beginning at day 7 in
C3 cells and at day 14 in F6 control cells.
Figure 9: Results of scanning densitometric measurement of osteocalcin steady-state mRNA in four minigene expressing (open symbols) and nonexpressing (closed symbols) clones of MC3T3-E1 cells. There were easily apparent differences at days 14 and 21. By day 28, consistent differences in steady-state levels of osteocalcin mRNA between minigene-expressing and nonexpressing clones were less apparent.
Expression of a dominant negative type I collagen mutation in the murine calvarial osteoblast cell line MC3T3-E1 resulted in deposition of a type I collagen matrix that has relatively high rates of turnover compared with control cultures. Deposition of the abnormal matrix resulted in suppression of one marker of osteoblast differentiation (alkaline phosphatase activity) and a significant increase in another (osteocalcin secretion). There was no compensatory up-regulation of the expression of other bone matrix proteins including type I collagen, decorin, bone sialoprotein, and osteonectin.
The
human pro-1(I) minigene product expressed by the pMG155 expression
plasmid contains an intact carboxyl propeptide required for molecular
assembly and 23 uninterrupted Gly-X-Y triplets of the
adjacent triple helical domain; it would therefore be expected to
co-assemble with wild-type mouse pro-
chains, and
SDS-polyacrylamide gel electrophoresis under nonreducing conditions
showed that the shortened pro-
1(I) chains were not secreted as
monomers (data not shown). The turnover of
[
H]hydroxyproline from the collagen matrix is due
primarily to loss of type I collagen molecules having either one or
both wild-type pro-
1(I) chains, since there are very few Y-positioned prolines available for prolyl hydroxylation in
the truncated triple helix of the minigene product(18) .
Furthermore, the increased matrix turnover may be enhanced by
alterations in matrix metalloproteinase production by cells producing
the minigene product. The reason why the collagenous matrix deposition
by 21 days is not much lower in cells secreting the truncated
pro-
1(I) chains is not entirely clear but may be due to the fact
that there were higher cell numbers in the minigene-expressing clones
that were measured for hydroxyproline deposition compared with the
control clones.
These experiments, which use a dominant negative type I collagen mutation to effect a targeted disruption of collagen fibrillogenesis, augment previous in vitro studies of collagen-osteoblast interaction in two ways. First, they lend support to the theory that suppression of osteoblast characteristics by ascorbate deprivation (2, 4, 6, 9) or by the addition of proline analogs (4, 9) is due at least in part to the inhibition of collagen deposition. Second, these data indicate that the increase in alkaline phosphatase activity associated with osteoblast differentiation depends not only on the overall quantity of collagen deposited by also on normal collagen structure.
An unanticipated
result of these studies was that the relative instability and abnormal
structure of the extracellular matrix in cells expressing the
pro-1(I) minigene was associated with a dramatic increase in
osteocalcin gene expression, particularly in earlier stages of cellular
differentiation. This elevation of osteocalcin expression was in
contrast to what was observed in studies in which collagen deposition
was merely reduced in quantity by ascorbate deprivation or by collagen
synthesis inhibitors; in the latter cases, both alkaline phosphatase
activity and osteocalcin gene expression failed to increase during
prolonged osteoblast cultures(4, 9) .
The
stimulation of osteocalcin gene expression in the presence of a rapid
matrix turnover suggests a more complex mechanism for its regulation.
Basal expression of osteocalcin associated with cellular
differentiation (33, 34) is detectable after about 14
days in MC3T3-E1 cells cultured under differentiating conditions (Fig. 8B); in cells that expressed truncated
pro-1(I) chains, osteocalcin gene expression began at day 7,
shortly after collagen deposition is detectable but before the expected
onset of basal expression that is associated with cellular
differentiation. It is also evident that the up-regulation of
osteocalcin expression occurred through a mechanism distinct from that
which is initiated by pharmacological doses of
1,25-dihydroxycholicalciferol (vitamin D); however, our experiments do
not exclude the possibility that trace amounts of vitamin D present in
bovine serum had a permissive effect on osteocalcin expression in cells
making an abnormal matrix.
It appeared that the osteocalcin gene
responded differently to high rates of turnover than the genes coding
for other components of the extracellular matrix (reviewed in (35) ). For example, type I collagen, decorin, and osteonectin
were not increased, even in longer cultures. Osteocalcin differs from
those matrix components in that it is thought to play a role in bone
turnover(36, 37, 38) . The data contained in
this report suggest that osteocalcin may be regulated by factors
released from osteoblast matrix undergoing high rates of turnover. The
identity of that factor or factors, the signal pathway that might be
used, and target sequences on the osteocalcin promoter are the objects
of further study. It is interesting to note that the abnormal matrix
did not induce an increase in the secretion of bone sialoprotein, an
approximately 80-kDa protein found in skeletal tissues. Its function is
not known, but it has been hypothesized to play a role in attachment of
osteoclasts to bone because it contains an RGD sequence and supports
the attachment of osteoclasts in vitro, perhaps through
ligation of the integrin(39) .
The mechanism by which deposition of a collagen-rich osteoblast matrix facilitates the transition from immature, rapidly dividing preosteoblasts to differentiated cells is unknown. Type I collagen may act directly on cellular receptors, or it might be merely required as a latticework for other matrix proteins which themselves interact directly with cells. It is unclear whether signals initiated by insoluble matrix molecules act directly to promote osteoblast-specific gene expression or whether cell-matrix interactions have indirect effects by which they might change in the repertoire of cellular receptors for hormones or cytokines present in the extracellular matrix (40, 41) . These studies suggest a need for better understanding of extracellular matrix receptors and downstream signaling pathways by which matrix components modulate gene expression in differentiating osteoblasts.
Although stable incorporation of informative genes in MC3T3-E1 cells is a powerful means to alter the extracellular environment of osteoblastic precursor cells, the clonal selection required for this strategy to work allows for the possibility that observed differences in osteoblast characteristics may be due to clonal variation rather than a response to the altered extracellular matrix.
In these studies, several steps
were taken to minimize the effect of clonal drift. All control clones
were chosen from cells that had been transfected with both pCDNA3 and
pMG155 and had undergone clonal selection with G418 but did not express
the pro-1(I) minigene. Second, all clones undergoing selection
were ``passed'' by brief trypsinization in the dishes twice a
week to maintain characteristics of differentiated osteoblasts.
Finally, several minigene-expressing and nonexpressing clones were
examined to confirm that discordant expression of osteocalcin and
alkaline phosphatase was a characteristic of clones that expressed the
pro-
1(I) minigene.