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| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cell Growth & Differentiation |
Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140 [G. R. B., E. M.], and CollaGenex Pharmaceuticals, Inc., Newtown, Pennsylvania 18940 [B. Z.]
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Abstract |
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 TOP Abstract IntroductionResults and DiscussionMaterials and MethodsReferences |
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B activator, cellular
retinoic acid-binding protein II (CRABP-II), and the transcription
factors S-II, SP2, and SEF2 (ITF2/E2:2). SEF2 is the first basic
helix-loop-helix protein found to be up-regulated during osteoblast
differentiation. Northern blots confirm the induction of SEF2. |
Introduction |
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TOPAbstractIntroductionResults and DiscussionMaterials and MethodsReferences |
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We used a mouse cDNA expression array containing 588 cDNAs to probe the changes induced in ascorbic acid-treated MC3T3-E1 cells. These cDNAs were selected on the basis that they represent genes reported to play key roles in many different biological processes, and they are each characterized by tight transcriptional regulation. The array includes numerous growth factors, cytokines, interleukins, and their receptors, as well as key genes involved in different stages of embryonic development. MC3T3-E1 cells (1) are a newborn mouse calvarial-derived cell line capable of differentiating along the osteoblast lineage (2, 3, 4) . MC3T3-E1 cells are an established clonal line, but they maintain much of the tightly linked controls between proliferation and differentiation usually seen only in primary cells (2) . These cells thus provide an excellent and frequently used model for studying patterns of gene expression in differentiating osteoblasts.
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Results and Discussion |
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TOPAbstractIntroductionResults and DiscussionMaterials and MethodsReferences |
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. The array is organized into six grids. In general, the cDNAs in grids
A, B, and C are designed to analyze the expression of genes related to
cell growth, whereas the cDNAs in grids D, E, and F analyze
tissue-specific gene expression. A complete list of the genes assayed
is shown in Table 1
. Noninduced MC3T3-E1
cells were originally compared with NIH3T3 cells to obtain a baseline picture of
gene expression in the calvarial line. Changes in gene expression
during differentiation were then probed in MC3T3-E1 cells
differentiating in response to induction with ascorbic acid. To get a
profile of gene expression during all phases of differentiation
(proliferation, matrix accumulation, and terminal differentiation
indicated by mineralization), RNA was harvested for analysis at days 0
(cells at 90% confluence), 3, 7, 14, and 21 after the addition of
differentiation medium. These points cover the various phases of
osteoblast differentiation and correlate with the time points at which
the expression patterns of osteoblast markers have typically been
studied. A time line illustrating the major phases of osteoblast
differentiation is shown in Fig. 2
A. Northern blot surveys of genes whose expression is known to be
regulated during osteoblast differentiation are shown in Fig. 2
B. The changing gene expression profiles indicate the
differentiation state of the cells at the approximate time points
chosen for the gene array analysis. The cells are actively replicating
at the 0 time point, as indicated by expression of histone H4. Histone
expression declines by day 4 and is almost undetectable by day 9.
Collagen type I is induced early, and its expression declines by day 14
as the mineralization phase nears completion. Osteocalcin is very
tightly regulated; its appearance correlates with transition to the
mineralization phase. Additional data on the state of the cells under
the conditions analyzed here can be seen in Refs. 5
and 6
.
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DNA, and pUC18. These were negative for all blots analyzed. The
blots also contain nine positive controls, which were averaged to
create a normalization value for each blot. Final values were
multiplied by a factor of 100 for ease of analysis. The normalized
results for both cell lines and all time points are shown in Table 1
.
Gene Expression in MC3T3-E1 Preosteoblasts Compared with NIH3T3
Cells.
The MC3T3-E1 line is derived from calvarial cells, hence these cells
are expected to display a level of tissue-specific gene expression even
before further differentiation. Expression patterns specific to the
MC3T3-E1 line compared with NIH3T3 cells can be derived from inspection
of columns 1 and 2 in Table 1
. To facilitate this comparison, genes
that show at least a moderate level of expression and vary by more than
2.5-fold between the two lines are listed in Table 2
. For the purposes of this study, a moderate level of expression was
considered to be a value greater than 20 in Table 1
. These limits are
arbitrary, but they allow the data to be reduced to a clearer picture
of the most notable differences in gene expression. The gene array is
designed to probe expression of genes that are active only under
specific conditions. Only about 35–40 of the 588 genes screened are
expressed at a moderate or higher level in NIH3T3 cells. Expression of
55–60 genes is apparent in MC3T3-E1 cells at day 0. The general
pattern is that additional genes are activated in MC3T3-E1 cells, but
there are both positive and negative differences between the two cell
lines. Some of these are striking. For example, expression of the
adipocyte differentiation-associated protein Pref-1 (Table 2
, line 35)
is undetectable in MC3T3-E1 cells, although it is expressed abundantly
in NIH3T3 cells. In contrast, expression of the CD44 cell surface
antigen (Table 2
, line 29) is not detectable in normal NIH3T3
fibroblasts but is detectable in MC3T3-E1 cells at all time points.
This is consistent with histology studies indicating that CD44 is
expressed in osteoblasts at all stages of maturation (7)
.
Gene array autoradiograms illustrating some of the differences between
expression in MC3T3-E1 cells and NIH3T3 cells are shown in Fig. 3
A. As an initial check on whether the gene array results are
representative of what would be revealed by Northern probes, we used a
Northern blot to analyze expression of Pref-1 and CD44 in the two cell
lines. The results (Fig. 3B)
confirm the reciprocal
differences between the two cell lines, both the striking difference in
expression level of Pref-1 and the more modest difference in expression
level of CD44.
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and 2
reveals differences in particular
classes of genes. MC3T3-E1 cells show a higher level of expression of
multiple genes associated with cell cycle activity. These include many
of the cyclins (grid locations A6a–A6l), and the Golgi
4-transmembrane-spanning transporter protein, which is associated with
nucleotide transport. Expression of several intracellular transduction
modulators (B2g–B7n) is also specifically active in MC3T3-E1 cells
relative to NIH3T3 cells. These include
Stat6,5
PKC-
, and the Rab-2 ras-related protein gene (Table 2
, lines 11, 14,
and 17). Several apoptosis-related proteins (C1a–C5d) are expressed at
relatively high levels in MC3T3-E1 cells, particularly Bcl-2 family
members (Table 2
, lines 19–21). The DNA synthesis, repair, and
recombination proteins (C5e–C7n) do not vary much as a class. MC3T3-E1
cells begin to withdraw from the cell cycle at about day 3 after they
are induced to differentiate, therefore it is expected that many of the
genes from this group will be down-regulated after ascorbic acid
treatment. Of the 21 genes from the A, B, and C grids that are
expressed at a 2.5-fold or higher level in the MC3T3-E1 cells relative
to the NIH3T3 cells, about half are sharply down-regulated when
MC3T3-E1 cells are induced with ascorbic acid. These are considered
further in Table 3
. Most of the others do not vary widely during differentiation. Only two
genes from the A, B, and C grids are specifically less active in
MC3T3-E1 cells. H-ras (Table 2
, line 22) was not detected in the
calvarial cells, in contrast to N-ras (A5e), which is slightly more
active in MC3T3-E1 cells. The gp130 signal-transducing subunit of the
interleukin 6 receptor is also expressed at a lower level in
MC3T3-E1 cells compared with NIH3T3 cells (Table 2
, line 23).
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, line 39). However, it is up-regulated more
than 3-fold in MC3T3-E1 cells at day 3 (Table 1)
, consistent with
suggestions that it plays a role in collagen synthesis
(8)
. Other differences are apparent in the table. Among
receptors and cell surface antigens (E1a–E7n), the granulocyte
macrophage colony-stimulating factor receptor is not expressed in the
calvarial cells (Table 2
, line 40). Calvarial cell-specific expression
of the CD44 antigen was noted above. The entire panel of 21
neurotransmitter receptors (E4e–E5k) is essentially not expressed in
either cell line or at any point during differentiation of the MC3T3-E1
cells. Because these genes are very specific to neuronal cells, they
are an excellent internal control. Among the growth factors, cytokines,
and chemokines, IGFBP-4 is specifically active in the calvarial cells
(Table 2
, line 31), whereas IGFBP-6 is specifically more active in
NIH3T3 cells (Table 2
, line 41), although the latter factor is induced
in the calvarial cells during differentiation. Some cytoskeletal
protein genes (F5e–F6e) appear to be more active in the calvarial
cells. The expression patterns of proteases and inhibitors (F6f–F7n)
are consistent with the matrix formation and remodeling functions of
calvarial cells. For example, expression of the serine protease
inhibitor homologue J6 (Hsp47; Table 2
, line 34) generally correlates
with expression of collagens and was previously reported to be induced
in MC3T3-E1 cells when collagen expression is induced (9)
.
Inspection of the entire data set in Table 1
reveals other differences
between the cell lines that were below the cut-off points chosen for
Table 2
. Though relatively small, many of these differences are likely
to be real and significant. For example, they include additional
differences among the matrix proteases.
Gene Expression in MC3T3-E1 Cells during Differentiation.
Differentiating MC3T3-E1 cells were analyzed at days 0, 3, 7, 14, and
21. Analyzing multiple time points gives a more active picture of
changes in gene expression over the course of the differentiation
process. However, the data are not easily presented as a simple list of
genes that are increased or decreased. Sample autoradiographs
illustrating results obtained at day 0 and day 21 are shown in Fig. 4
. A comprehensive picture can be derived from inspection of the full data
set in Table 1
. The most significant changes are discussed below.
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is higher
than 20 and show a change of at least 2.5-fold maintained over at least
two consecutive assay points are listed in Table 3
. An internal control
is illustrated through analysis of the cyclins. Ascorbic acid-induced
MC3T3-E1 cells exit the cell cycle between days 3 and 7 of the
differentiation process. If the expression array analysis is reflective
of known results, expression of A- and B-type cyclins should decrease
by day 7. Analysis of the cyclins included on the blots confirms this
time course. Cyclins B1 and B2 are included in Table 3
, but the data in
Table 1
indicate that cyclins A, B1, B2, and C all decrease between
days 0 and 7. The results are presented as a graph in Fig. 5
. As shown in Tables 1
and 2
, several additional cyclins are expressed at
relatively high levels in noninduced MC3T3-E1 cells. These include
cyclins D1, D2, and G. Expression of these genes is maintained at
relatively high levels throughout differentiation. Cyclin G can be
induced in neuronal cells in response to BMP-4 and may play a role in
the modulation of apoptosis (10)
. Presumably, it plays a
significant role in osteoblast differentiation as well.
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is p53. The ability of p53 to control and
coordinate many aspects of proliferation and apoptosis in response to
various internal and external stimuli has been well studied. Regulation
of p53 expression during differentiation is not generally expected.
Nevertheless, the expression array indicates that p53 expression is
relatively high in noninduced MC3T3-E1 cells and that it decreases
markedly by day 7 of differentiation. The hybridization results for p53
at all time points are shown in Fig. 6A
and plotted in Fig. 6
B. To probe the extent to which the
data from the expression array reflect synthesis of the protein
product, p53 was analyzed at the level of protein synthesis in
differentiating osteoblasts. The results of the immune precipitation
(Fig. 6C)
were consistent with the pattern of RNA expression
indicated in the gene array analysis. These results reveal an
unexpected but potentially important role for regulation of p53
expression in calvarial cells.
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reveals many additional changes that may also be significant. These
include changes in genes that are expressed at low levels or that vary
less than 2.5-fold or are down-regulated at a single time point.
Cyclins A and C are examples. Other patterns are also apparent. The
STAT genes (B4d–B4g) are generally down-regulated, whereas other genes
of this type are activated (B4h–B4j). Various changes are apparent
within the intracellular kinase and G protein networks (B5f–B7n).
Small changes are apparent in various apoptosis-related proteins
(C1a–C5d). In general, as expected for cells withdrawing from the cell
cycle, the genes that are down-regulated are from the classes
represented by the A, B, and C grids, whereas genes that are
up-regulated are from the D, E, and F grids. The major exception among
the transcription factors is the repression of the multiple homeodomain
protein gene ATBF1 (Table 3
, line 9). Another transcription
factor, butyrate response factor 1 (D1i), is consistently
down-regulated, but not within the parameters chosen for Table 3
.
Genes That Are Up-Regulated during Osteoblast Differentiation.
Inspection of the data in Table 1
indicates that the time of peak
expression for the genes induced during differentiation can vary. Genes
that reach a peak expression level of at least 20 and show at least a
2.5-fold increase in expression maintained over at least two
consecutive time points are listed in Table 4
. In general, genes that are induced tend to be various transcription
factor-related genes, growth factor-related genes, intracellular
signaling protein genes, and antiapoptosis protein genes. Many other
genes are induced at a lower level. These can be identified by
inspection of the full data set in Table 1
. These genes tend to be from
the same classes, with the addition of some cell growth inhibitors such
as p21 (A7e) and p27 (A7f). A few factors associated with cell growth
are activated. These include VEGF receptor 1 (A4j), the Lfc
proto-oncogene (A5d), Shc (A5f), and transcription factor TFIID (B4j).
Known targets of p53 are up-regulated as the cells withdraw from the
cell cycle, although p53 is down-regulated. However, p53 expression in
the calvarial cells is initially high and is never significantly
reduced below the level seen, for example, in NIH3T3 cells. Table 4
highlights changes that reflect withdrawal from the cell cycle and the
subsequent activation of specific growth factors, receptors, and
transcription factors. Some of these are expected; others have not
previously been reported in osteoblasts.
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, line 1) is not detected in noninduced MC3T3-E1 cells. Its
activation in differentiating osteoblasts has not been reported
previously, but it is strongly activated with a peak expression at day
3, which suggests that it plays a significant role in the
down-regulation of proliferation as the cells proceed to the
mineralization phase. Transcription factor A10 (Table 4
, line 5), also
designated EKLF, is known as an erythroid cell-specific transcription
factor (13
, 14)
, thus its induction in differentiating
osteoblasts is also unexpected. However, the strong induction and peak
expression at day 14, when osteogenic proteins are most actively
transcribed, suggests that this factor may play a significant role in
osteoblast-specific gene expression.
EI24 (Table 4
, line 2) was isolated as a gene product induced in a
p53-dependent manner in NIH3T3 cells treated with the cytotoxic drug
etoposide (15)
. It is also induced by ionizing radiation.
Its induction during normal differentiation in osteoblasts has not been
reported, but it is interesting that its expression in osteoblasts
roughly parallels the unusually high expression of p53. Gadd45 (Table 4
, line 6) is also strongly induced at later stages of differentiation.
Like EI24, this product is usually regulated by p53 in response to DNA
damage. An advantage of the gene array approach is its potential to
reveal such unexpected patterns as the induction of these specific DNA
damage response genes during normal differentiation.
The oxidative stress-induced protein A170 has been studied in
macrophages (e.g., Ref. 16
) and was recently cloned as a
gene up-regulated in MC3T3-E1 after treatment with TGF-ß
(17)
. The gene array analysis reveals that it is also
strongly activated in response to ascorbic acid induction (Table 4
,
line 3). Activation is apparent by day 14 and persists through the
mineralization phase. The transcription factor Nrf2 (Table 4
, line 9)
regulates a wide-ranging metabolic response to oxidative stress,
including activation of A170 (see, for example, Ref. 18
). The
activation of Nrf2 in combination with A170 implies that an oxidative
stress response is a regulated aspect of osteoblast differentiation in
culture, most likely as a consequence of matrix accumulation and
mineralization. This may relate to the critical requirement for
angiogenesis during bone growth and fracture healing. VEGF (F4j) is
strongly linked with angiogenesis and is expressed in mouse osteoblasts
at late stages of differentiation (19)
. It can also be
induced in an osteoblast-like cell line in response to hypoxia
(20)
. VEGF induction at the RNA level is just barely
detectable on the blot at day 21 (F4j). However, induction of the VEGF
receptor (A4j) is more clearly apparent in Table 1
, although it is
below the cut-off chosen for Table 4
.
Induction of a member of the TRAF family of NF-B activators in
differentiating osteoblasts (Table 4
, line 4) is interesting in light
of a report suggesting that TRAF family members play a role in the
regulation of osteoclast function (21)
. Expression of
stromelysin-3 (Table 4
, line 7) has been observed in MC3T3-E1 cells
(22)
, but its specific induction during differentiation in
response to ascorbic acid has not been reported previously.
CRABP-II (Table 4
, line 9) is presumed to modulate the level of
retinoic acid available to bind to the receptor in cells. Expression of
CRABP-II has been observed only in selective tissues and cell types;
these include human osteoblasts (23)
. The gene is
expressed at a very low level in noninduced MC3T3-E1 cells, but it is
activated 4–10 fold as the cells progress to a more osteoblast-like
phenotype. The usual effector for CRABP-II expression is retinoic acid,
and expression of CRABP-II is generally associated with coexpression of
the retinoic acid nuclear receptors (retinoic acid receptors and RXRs).
Expression of RXR-
(D6f), ß2-retinoic acid receptor (B3k), or
RXR-ß cis-11 (B4c) was not detected on the blots, although
other forms of the receptor may be expressed. The retinoid-related
orphan receptor (ROR)-
1 (D5i) is induced by day 7. This is
consistent with recent results indicating a significant role for the
ROR- receptor in bone metabolism (24)
. Another
component of this signaling pathway, the RXR interacting protein
(RIP-15; D6g), is induced with kinetics similar to CRABP-II, although
the expression level is lower. CRABP-II expression can be stimulated by
TGF-ß in mouse embryonic palatal cells (25)
. Consistent
with this finding, CRABP-II expression parallels an increased
expression of TGF-ß (Table 4
, lines 18 and 19; discussed further
below) in the MC3T3-E1 cells.
IGFs have been shown to enhance matrix formation and cell proliferation
in bone models (reviewed in Ref. 26
). The IGFs are modulated by the
binding of IGFBPs (27)
. The expression of IGFBP-5 and
IGFBP-6 (Table 4
, lines 17 and 18) in MC3T3-E1 cells during
differentiation is expected and is consistent with the activities of
these proteins in modulating the effects of IGF-1 (F3a) and IGF-2
(F2n). Expression of many of these factors in MC3T3-E1 cells has been
described previously (28)
, although these investigators
did not analyze expression through each stage of differentiation. The
peak expression of IGFBP-6 and IGFBP-5 at about day 14 is consistent
with a previous study (29)
using MC3T3-E1 cells. In
contrast to that report, the expression array did not show a marked
increase in expression of IGFBP-2 (A5m). We did, however, observe an
increase in expression of IGFBP-4 (F2l), although its expression was
fairly high even before exposure to differentiation medium, as
discussed under Table 2
. IRSs (IRS-1 and IRS-2) are critical for
mediating the anabolic effects of insulin and IGF-1 (F3a) on bone
metabolism (30)
. The blots indicate expression of IRS-1 in
MC3T3-E1 cells before differentiation and an increase of more than
5-fold by day 14 of differentiation (Table 4
, line 14). As indicated
under Table 2
, IRS-1 was not detected in NIH3T3 cells. Consistent with
its role in IGF signal transduction, we found that the increase in
IRS-1 expression in MC3T3-E1 cells parallels that of the IGFBPs,
peaking at day 14.
The TGF-ß superfamily consists of TGF-ßs, BMPs, and activins. They
are multifunctional proteins that exert their biological effects by
signaling through a family of heteromeric serine/threonine kinase
receptors (reviewed in Ref. 31
). TGF-ß1 and TGF-ß2 are associated
with the extracellular matrix and are integral players in the processes
of bone formation (32)
. The increased expression of
TGF-ß1 and TGF-ß2 (Table 4
, lines 18 and 19) and BMP-2 and BMP-4
(discussed below) during differentiation of the MC3T3-E1 cells is
consistent with an important role for the TGF-ß superfamily in bone
cell development and remodeling.
BMP receptor 1A (Table 4
, line 13) is a serine/threonine kinase
receptor that mediates the osteogenic effects of the BMPs. It is
specifically expressed in the calvarial cells relative to NIH3T3 cells
and is activated as much as 10-fold during differentiation of MC3T3-E1
cells. The kinetics of its expression, peaking at day 14 and declining
by day 21, when mineralization is apparent, correlate with the
expression pattern of many of the other osteogenic proteins discussed
here. The constitutive expression of the BMP-2 and BMP-4 ligands in
MC3T3-E1 cells has been reported previously (33)
. These
authors found that expression of functional BMP receptor 1A is required
for progression of these cells to a mineralization phenotype during
long-term culture. Expression of several members of the BMP family
(F1a–F1f) is detectable, as shown in Table 1
. The temporal expression
pattern of BMP receptor 1A is a potential mechanism to coordinate the
osteogenic effects of the BMPs with other differentiation mediators.
Follistatin (Table 4
, line 15) is an activin-A-binding protein.
Activin-A, a member of the TGF-ß superfamily, is a pluripotent growth
factor with important roles in development, erythropoiesis, and the
local regulation of many tissues. Hashimoto et al.
(34)
reported that MC3T3-E1 cells contain a high number of
activin-binding sites on their surface and that activin promotes
mitogenesis and suppresses alkaline phosphatase activity in these
cells. Activin activity is suppressed through the formation of
complexes with inhibin and follistatin. The inhibin subunit (F2g)
is detectable only at very low levels on the blots. The inhibin ßA
subunit (F2h) is expressed at higher levels and is induced with the
same kinetics as follistatin, but just below the cut-off point chosen
for Table 4
. The kinetics of follistatin and inhibin ßA expression
observed here are consistent with the timing of alkaline phosphatase
induction in MC3T3-E1 cells after exposure to ascorbic acid
(5)
. The blots do not show consistent expression of the
activin type I receptor (E1a), although other activin-binding sites may
be present on these cells.
Cathepsins are secreted lysosomal proteases that mediate the
degradation of certain cellular and extracellular proteins, inside and
outside of the lysosome. Cathepsin K has been demonstrated to play an
important role in osteoclast function because of its ability to
efficiently degrade type-1 collagen (reviewed in Ref. 35
), but
induction of cathepsins has not been studied previously during ascorbic
acid-mediated differentiation in osteoblasts. This family also
proteolytically activates proenzymes, prohormones, and growth factors.
Activation of TGF-ß in human osteoblasts in response to
glucocorticoid treatment correlates with a dose-dependent increase in
the mRNA levels of cathepsin B and D (36)
. Inspection of
Table 1
indicates that cathepsin B (F6g) and cathepsin D (F6h; Table 4
,
line 20) are also induced at certain times in ascorbic acid-treated
MC3T3-E1 cells. The expression arrays support the suggestion that
increased expression of the cathepsins is an important factor in the
proteolytic activation of TGF-ß in differentiating osteoblasts and
possibly in remodeling of the matrix.
The D grid of the expression array presents cDNAs for almost 100
tightly regulated transcription factors. The Cbfa1 transcription
factor, which is not included on the array, is clearly associated with
osteoblast-specific gene expression (reviewed in Refs. 37
–39).
However, the transcription factor profile in differentiating cells is
complex, and the expression array affords an opportunity to compare the
relative expression of numerous factors of interest. In addition to
Nrf2, discussed above, three other transcription factors are activated
above the limits set for Table 4
. These are transcription factors S-II,
SEF-2, and SP2 (Table 4
, lines 10–12). None of these has been studied
in osteoblasts before. The expression pattern of all three factors
mimics that of the typical differentiation factors, starting out low,
increasing until day 14, and then decreasing after the onset of
mineralization. Transcription factor S-II (TFIIS) is a member of a set
of general transcription elongation factors that permit RNA polymerase
II to transcribe faster and more efficiently (reviewed in Ref. 40
). In
vertebrates, a family of related genes has been identified, of which
some members are expressed in a tissue-specific manner. The arrays
indicate that expression of TFIIS is restricted to a point in
osteoblast differentiation at which proliferation has ceased, and the
expression of genes associated with differentiation has been initiated.
Transcription factor SP2 is part of the SP1 multigene family. It has
been reported to play a role in the regulated expression of the T-cell
receptor and the CT genes (41
, 42)
.
Transcription factor SEF2 (also known as ITF2 or E2-2) is a member of the E factor class of the bHLH transcription factor family. Almost every member of this family has been implicated in the regulation of transcription during cell type determination and differentiation. Several laboratories have examined the role of bHLH proteins in osteoblasts. Functional E boxes have been demonstrated in osteocalcin promoter assays in MC3T3-E1 cells and other osteoblast-like cell lines (43, 44, 45) . Two recent studies report a negative role for the bHLH proteins TWIST and DERMO-1 in osteoblast differentiation. Overexpression of TWIST causes dedifferentiation in the human osteosarcoma line SaOS2, whereas underexpression leads to up-regulation of osteoblast markers including alkaline phosphatase, type I collagen, and osteopontin (46) . Tamura and Noda (47) demonstrated expression of DERMO-1 mRNA in undifferentiated MC3T3-E1 cells and found that it is expressed at a lower level in these cells by day 21 of differentiation. The results presented here represent the first report of SEF2 expression in osteoblasts. The time course of SEF2 expression suggests that this bHLH protein plays a positive role in transcriptional regulation of the osteoblast phenotype.
Inspection of the raw data in Table 1
reveals many additional changes
that may also be significant, although they involve lower expression
levels or induction at only a single assay point. The activation of the
Y box-binding protein (D7j) at day 3 was discussed under Table 2
.
IFN-regulatory factor 2 (D4l) is activated, whereas IFN-regulatory
factor 1 is repressed (B7k in Table 3
). IFN-inducible protein 1 (D4k)
is induced. The glucocorticoid receptor form A (E3m), growth hormone
receptor (E3n), and insulin receptors (E4a) are induced at low but
fairly consistent levels. In contrast, the androgen (E3j), calcitonin
(E3k), and estrogen (E3l) receptors show no expression at any assay
point.
Many of the observed changes, such as the induction of follistatin, BMP
receptor 1A, TGF-ß, and matrix remodeling factors, reflect expected
patterns and support the physiological relevance of the results. Other
observed changes were not anticipated and offer new insight into the
osteoblast differentiation process. To evaluate the reliability of the
data indicating unexpected gene inductions, we performed Northern blot
analyses on selected genes with distinct expression patterns. The genes
we chose include SEF2, because this is the first bHLH protein found to
be up-regulated during osteoblast differentiation. We also examined the
oxidative stress-associated proteins A170 and Nrf2. The results of the
Northern blots are shown in Fig. 7
, where they are compared with the gene array results depicted in
bar graphs. The gene array analysis indicated a steady rise
in expression of SEF2, with a peak at about day 14. The Northern blots
show an almost identical pattern. A170 was essentially undetectable in
the gene array analysis until it rose sharply at about day 14. The gene
arrays suggest that A170 expression falls off sharply as well. The
Northern blots confirm this dramatic rise and fall in expression. The
gene arrays indicate that Nrf2 is induced 4–5 fold by day 7 and
remains fairly high thereafter. The Northern blot is mostly consistent
with this pattern. The Northern blot suggests that induction may begin
by day 4. The Nrf2 signal on the Northern blots does not stay
consistently elevated, but this may represent normal variation in
assays. Overall, the Northern blots give a high degree of confidence
that the results from the gene array are a fair representation of
actual changes in gene expression during osteoblast differentiation.
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Summary.
The data generated from this type of approach are largely descriptive.
However, the overview that is obtained is a very useful baseline for
more analytical probes of functional pathways. Differentiation in any
system involves a cascade of signals from receptor-mediated events to
transcription factor-mediated changes in gene expression. The analysis
presented here offers a dynamic picture of these events during
osteoblast differentiation. In this context, it is important to note
not just the genes that change but also those that are not expressed or
induced. Where comparisons are possible, the expression array results
presented here are generally consistent with previous studies. However,
many genes previously studied in osteoblasts were analyzed under
different conditions, in different cell lines, or under induction with
different agents. The ability to compare data collected on hundreds of
genes under one set of conditions with data from other systems
strengthens our overall understanding of the molecular basis of
osteogenesis. The analysis has also produced several novel insights, as
discussed throughout the text. These data are valuable not just for a
better understanding of osteogenesis but also for the comparisons they
permit with other tissue types. This information should aid in the
development of effective treatments for bone disorders. It may also
help predict side effects on bone metabolism from drugs that target the
same factors for intervention in other diseases.
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Materials and Methods |
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TOPAbstractIntroductionResults and DiscussionMaterials and MethodsReferences |
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-MEM (Irvine
Scientific) plus 10% fetal bovine serum (Summit Biotechnologies),
supplemented with 50 units/ml penicillin and 50 µg/ml streptomycin
(Mediatech). For differentiation assays, cells were plated at an
approximate initial density of 5 x 104
cells/cm2. Differentiation was induced by the addition of
50 µg/ml (final concentration) ascorbic acid (Sigma) and 10
mM (final concentration) ß-glycerol phosphate (Sigma) to
standard growth medium. The medium was changed every 3–4 days, and the
inducing agents were replaced with each media change. NIH3T3 cells were
a gift from Scott Shore (Temple University, Philadelphia, PA). These
cells were grown in DMEM (CellGro) plus 10% fetal bovine supplemented
with 50 units/ml penicillin and 50 µg/ml streptomycin, as described
above.
cDNA Array.
We used the Atlas mouse cDNA Expression Array (Clontech 7741-1) and the
recommended protocol. The internet support
site6
maintained by the manufacturer includes a complete list of genes
in the array with a short description of gene function and a list of
relevant publications.
RNA and Gene Array Probe Preparation.
Total cell RNA was prepared from appropriate cell cultures at various
time points using Trizol reagent (Life Technologies, Inc.) according to
the manufacturer’s recommendations. Samples were treated with DNase
(Boehringer Mannheim) before probe preparation. Labeled probes were
prepared by reverse transcription of 5 µg of RNA in the presence of
[32P]dATP (New England Nuclear). Probes were hybridized
to the membrane overnight, the membrane was washed thoroughly according
to the manufacturer’s recommended protocol, and the results were
visualized by either a PhosphorImager for quantification or by
autoradiography. Signals were quantitated by exposure on a Fujix BAS
200 (Fuji) and analyzed with MacBAS 2.0 software. The blots contain
nine negative controls and nine positive controls. The positive
controls can be used with phosphorimaging to normalize the signal from
blot to blot.
cDNA Northern Probes.
cDNA probes, type-I collagen, osteocalcin, and actin have been
described previously (5)
. The cDNA for histone H4 was
purchased from American Type Culture Collection (clone ID, 775450).
cDNAs used for PREF-1, SEF-2, A170, CD44, and Nrf2 were created by
reverse transcription-PCR. Primer sequences were purchased from
Clontech.
Northern Blots.
Total cell RNA was prepared from appropriate cell cultures at various
time points using Trizol reagent (Life Technologies, Inc.) according to
the manufacturer’s recommendations. Twenty µg of RNA were loaded per
lane and fractionated by electrophoresis through a 1%
formaldehyde-agarose gel. The RNA was transferred to a Hybond-N nylon
membrane (Amersham Life Science Inc.) and cross-linked by UV
irradiation and baking at 80°C. 32P-labeled probes were
prepared using a random primed labeling kit (Boehringer Mannheim).
Between successive probes, blots were stripped by treatment with
boiling in 0.1% SDS.
Radiolabeling Cellular Proteins and Immunoprecipitation.
MC3T3-E1 cells were incubated with
[35S]methionine/cysteine (New England Nuclear) in
methionine/cysteine-free DMEM (Life Technologies, Inc.) for 2 h.
Cells were washed in PBS and collected by centrifugation (2000 rpm for
5 min). Cell pellets were lysed in p300 lysis buffer (49)
supplemented with the following protease inhibitors at a final
concentration as indicated: aprotinin (2.0 µg/ml); leupeptin (2.0
µg/ml); and pepstatin (1.0 µg/ml). One mg of total cell lysate
(Bradford assay) was immunoprecipitated with the p53 monoclonal
antibody PAb421 (50)
. The immunoprecipitation was
separated by 10% SDS-PAGE and visualized by autoradiography.
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Acknowledgments |
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Footnotes |
|---|
1 Supported by National Cancer Institute Grant
CA53592 (to E. M.), a biomedical science grant from the Arthritis
Foundation (to E. M.), and the Temple University Research
Enterprise Program (E. M.). 
2 G. R. B. was supported in part by NIH
Training Grant T30 CA09214, by a Daniel Swern Fellowship from Temple
University, and by National Cancer Institute Scholar Award CA84573.
3 Present address: National Cancer Institute, Gene
Regulation Section, Building 560, Room 21-21, Frederick, MD
21702-1201.
4 To whom requests for reprints should be
addressed at Fels Institute for Cancer Research and Molecular Biology,
Temple University School of Medicine, Philadelphia, PA 19140. Phone:
(215) 707-7313; Fax: (215) 707-6989; E-mail: betty{at}unix.temple.edu
5 The abbreviations used are: STAT, signal
transducers and activators of transcription; IGFBP, insulin-like growth
factor-binding protein; VEGF, vascular endothelial growth factor; TGF,
transforming growth factor; Nrf2, NFE2-related factor 2; RXR, retinoid
X receptor; IGF, insulin-like growth factor; IRS, insulin receptor
substrate; BMP, bone morphogenetic protein; bHLH, basic
helix-loop-helix; SAGE, serial analysis of gene expression; PKC,
protein kinase C; NF-B, nuclear factor B; CRABP, cellular
retinoic acid-binding protein; TFII, transcription factor II; TRAF,
tumor necrosis factor receptor-associated factor.
6 Internet address:
http://www.clontech.com/archive/JAN98UPD/Atlaslist.html
Received for publication 9/ 6/00. Revision received 12/15/00. Accepted for publication 12/18/00.
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References |
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TOPAbstractIntroductionResults and DiscussionMaterials and MethodsReferences |
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), B1
(
), B2 (
), and C (•) genes.
