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J Biol Chem, Vol. 275, Issue 7, 5037-5042, February 18, 2000
From the Department of Pharmacological and Physiological Science,
St. Louis University School of Medicine,
St. Louis, Missouri 63104
Previously we showed that the activator protein-1
site and the runt domain binding site in the collagenase-3
promoter act cooperatively in response to parathyroid hormone (PTH) in
the rat osteoblastic osteosarcoma cell line, UMR 106-01. Our results of
the expression pattern of core binding factor The predominant targets of parathyroid hormone
(PTH)1 are the skeleton and
kidney. PTH binds to PTH/PTH-related protein receptors present on the
plasma membrane of osteoblasts (1) and induces the expression of genes
such as collagenase-3 (matrix metalloproteinase-13) (2), interleukin-6,
leukemia inhibitory factor (3), insulin-like growth factor-I (4), and
prostaglandin G/H synthase-2 (5). Collagenase-3 is able to degrade
types I, II, III, and IV collagen, the cartilage proteoglycan,
aggrecan, and other components of the extracellular matrix (6-8).
Apparently due to its ability to degrade a wide range of matrix
components, its physiological expression is limited to situations in
which rapid and effective remodeling of collagenous extracellular
matrices is required, i.e. fetal bone development and
postnatal bone remodeling in both rodents (9, 10) and humans (11, 12).
By mutating the cleavage site for collagenase in type I collagen, it
has also been shown that this enzyme has a critical role in mediating
PTH-induced osteoclastic bone resorption in vivo (13).
Collagenase-3 is strongly induced by bone resorbing agents such as PTH
in the rat osteoblastic osteosarcoma cell line, UMR 106-01 (2, 14).
Previously we have identified the activator protein-1 (AP-1) and
runt domain (RD) binding sites as necessary for PTH-induced
collagenase-3 promoter activity. We have also demonstrated that there
is increased binding of c-Fos and c-Jun proteins to the AP-1 site in
response to PTH, whereas there is no change in the abundance of Cbfa
(AML)-related proteins binding to the RD binding site (15). Cbfa is a
mammalian homolog of the Drosophila genes runt
(16) and lozenge (17). Bone-specific genes such as
osteocalcin and osteopontin (major noncollagenous components of the
bone matrix) contain Cbfa consensus sites, referred to as the
polyomavirus enhancer-binding protein-2 Cbfa1 is essential for the maturation of osteoblasts, and targeted
disruption of the cbfa1 gene in mice produced skeletal defects (21, 22) that are essentially identical to those found in human
cleidocranial dysplasia. A recent study shows that the mutant mice do
not express collagenase-3 during fetal development, indicating that
collagenase-3 is one of the target genes regulated by Cbfa1 (23). Cbfa1
is also involved in osteoclastogenesis through regulation of osteoclast
differentiation factor/osteoprotegerin ligand in osteoblast lineage
cells (24). Cbfa1 is a key transcription factor in bone cells, and the
activity of runt proteins is required for completion of
osteoblast differentiation (25, 26). Cbfa1 is regulated by signaling
through Materials--
Parathyroid hormone (rat 1-34) was purchased
from Sigma. Restriction endonucleases were products of New England
BioLabs, Inc., Beverly, MA, and radionuclides were obtained from NEN
Life Science Products. Synthetic oligonucleotides were synthesized by
Life Technologies, Inc. Tissue culture media and reagents were obtained from the Washington University Tissue Culture Center, St. Louis, MO.
Fetal bovine serum was a product of JRH Biosciences, Lenexa, KS and was
also purchased through Washington University. All other chemicals were
obtained from Sigma or Fisher.
Western Blot--
UMR 106-01 cells were cultured as described
previously (15). Cell lysates containing 50 µg of total protein in
lysis buffer were electrophoresed by 12% SDS-PAGE. The proteins were
transferred electrophoretically to polyvinylidene difluoride membrane
(Bio-Rad). After blocking in Tween-Tris-buffered saline (0.1% Tween
20, 138 mM NaCl, 5 mM KCl, and 25 mM Tris-HCl (pH 8.0)) containing 5% (w/v) nonfat dry milk,
the membrane was probed with affinity-purified anti-Cbfa1 antibody
(diluted 1:1000) (anti-human AML-3, kindly provided by Dr. Scott
Hiebert) followed by incubation with horseradish peroxidase-conjugated
goat anti-rabbit secondary antibody (diluted 1:5000). The
antigen-antibody complexes were detected by ECL (Amersham Pharmacia Biotech).
Northern Blot--
Poly(A)+ RNA from control and
PTH-treated UMR cells was isolated using the FastTrack kit
(InVitrogen). Poly(A)+ RNA (1 µg/lane) was
electrophoresed on a 1% agarose, 2.2 M formaldehyde gel in
MOPS buffer (40 mM MOPS (pH 7.0), 10 mM sodium
acetate, and 1 mM EDTA). RNA was transferred to Zeta-Probe
GT-membrane (Bio-Rad) and hybridized in 50% formamide, 5X SSC, 10X
Denhardts, 0.1% SDS, 50 mM Na3PO4,
and 100 µg/ml salmon sperm DNA at 42 °C. Antisense RNA and
cDNA probes used for hybridization were labeled with riboprobe and
random priming kits (Promega), respectively. A mouse Cbfa1
(Osf2/Cbfa1)-specific cDNA was kindly provided by Dr. Gerard Karsenty.
DNA Transfection--
Earlier we characterized the rat
collagenase-3 promoter and identified the region conferring maximal PTH
responsiveness to within 148 base pairs upstream from the transcription
start site and the collagenase-3 promoter construct (
UMR cells were seeded in 6-well plates (2 × 105
cells/well) and transiently transfected with either 100 ng of
collagenase-3 promoter construct containing CAT as a reporter gene or
50 ng of Gal4 construct and 50 ng of reporter luciferase plasmid or the
amounts of DNA indicated in the figures using LipofectAMINE as
recommended by the manufacturer (Life Technologies, Inc.). PTH
treatment was performed the following day either for 24 h for CAT
reporter gene transfection or 6 h for luciferase reporter gene
transfection. CAT activity was measured in duplicate after the addition
of 25 or 50 µl of cell lysate to a 100-µl reaction volume
consisting of final concentrations of 250 µM
n-butyryl-coenzyme A and 23 mM
[14C]chloramphenicol (0.125 Ci/assay). Twenty µl of
cell lysate was used for measuring luciferase activity with a
luminometer, Optocomp II, using the luciferase assay system (Promega).
All experiments were repeated three times.
In Vitro Phosphorylation--
The recombinant wild type and PKA
mutant AD3 proteins were expressed from the pET30 vector in
Escherichia coli BL21 cells (Novagen). When the optical
density (600 nm) of the expression culture reached 0.4-0.6, expression
of the protein was induced by the addition of 0.1 mM
isopropyl- Recent work in this laboratory has identified two elements, namely
the AP-1 and RD sites, as required for collagenase-3 promoter activity
in response to PTH in the rat osteoblastic osteosarcoma cell line UMR
106-01 (15). We have also shown that there is an increased amount of
c-Fos and c-Jun proteins binding to the AP-1 site in response to PTH,
but there is no change in the abundance of Cbfa (AML) binding to the RD
site. Three isoforms of Cbfa1 have been identified that differ in their
N-terminal sequence (30). To identify whether there is a change in the
levels of Cbfa1 isomeric forms by PTH, total cellular lysates from
control and PTH-treated UMR cells were isolated and used for Western
blot analysis. Both control and PTH-treated lysates contained a major protein of ~45 kDa that was recognized by an affinity-purified antibody made against Cbfa1 (anti-human AML-3 raised against a C-terminal peptide) (Fig. 1A).
In addition, we have also found a minor protein of ~35 kDa. Since
there was no change in the abundance of the Cbfa1 isoforms during PTH
treatment, the possibility exists that either different Cbfa1 isoforms
of the same mass are expressed or, more likely, that Cbfa1 is
posttranslationally modified. The expression pattern of Cbfa1
transcripts in control and PTH-treated UMR cells was identified by
Northern blot analysis using a mouse Cbfa1-specific riboprobe. This
riboprobe hybridized to three transcripts (6.5, 5.4, and 3.2 kilobases)
in either control or PTH-treated UMR cells (Fig. 1B). There
was no change in the level of Cbfa1 RNA in either control or
PTH-treated conditions. The RNA loading and efficiency of transfer were
verified by reprobing the blot with Earlier, we showed that PTH phosphorylates CREB, resulting in
activation of the c-fos promoter (31). Recently, we also
reported that the PTH-induced phosphorylation of CREB is mediated by
the PKA pathway (32). Identifying the PTH signaling pathway for collagenase-3 promoter activity would provide insight into a possible posttranslational modification of Cbfa1. Hence, to identify the signaling pathway involved for PTH-induced collagenase-3 promoter activity, we used PKA, PKC, and ERK pathway inhibitors. The
collagenase-3 promoter construct (
Parathyroid Hormone Regulation of the Rat Collagenase-3
Promoter by Protein Kinase A-dependent Transactivation
of Core Binding Factor 
1*
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1 (Cbfa1), which binds
to the runt domain site, indicated that there is no change in the levels of Cbfa1 protein or RNA under either control conditions or after PTH treatment. The importance of posttranslational
modification of Cbfa1 in the signaling pathway for PTH-induced
collagenase-3 promoter activity was analyzed. PTH stimulation of
collagenase-3 promoter activity was completely abrogated by protein
kinase A (PKA) inhibition. To determine the role of PKA activity with
respect to Cbfa1 activation (in addition to its known activity of
phosphorylating cAMP-response element-binding protein to enhance
c-fos promoter activity), we utilized the heterologous Gal4
transcription system. PTH stimulated the transactivation of activation
domain-3 in Cbfa1 through the PKA site. In vitro
phosphorylation studies indicated that the PKA site in the wild type
activation domain-3 is a substrate for phosphorylation by PKA. Thus, we
demonstrate that PTH induces a PKA-dependent
transactivation of Cbfa1, and this transactivation is required for
collagenase-3 promoter activity in UMR cells.
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A/osteoblast specific
element-2/nuclear matrix protein-2 site (18-20).
2-integrin (27). The expression of Cbfa1 is also controlled
by Smad2, an essential intracellular component for the signal
transduction of transforming growth factor-
in osteoblastic cells
(28). Earlier, we demonstrated that only the level of c-Fos and c-Jun,
not Cbfa1, is increased by PTH, and even in the presence of c-Fos,
c-Jun, and Cbfa1, collagenase-3 promoter activity is strongly increased
by PTH treatment (15). These results suggest that a posttranslational
modification of Cbfa1 may be required for PTH induction of the
collagenase-3 gene through Cbfa1 activity. To understand the Cbfa1
regulation in PTH-induced collagenase-3 promoter activity, we analyzed
its expression by Western blot and Northern blot analyses. We dissected
the signaling pathway involved for PTH-induced collagenase-3 promoter
activity using ERK, PKC, and PKA inhibitors. Furthermore, the
posttranslational modification of Cbfa1 was determined using the
heterologous Gal4 transcription system. Cbfa1 was shown to be
phosphorylated in vitro by PKA. Our results provide evidence
of PKA-dependent Cbfa1 transactivation, specifically as
required for PTH-induced collagenase-3 promoter activity in UMR cells.
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148 C'ase-3)
contains both the AP-1 and RD sites (15). The wild type Gal4 AD3
construct, containing the activation domain-3 (AD3) in the proline-,
serine-, threonine-rich domain region of mouse Osf2/Cbfa1 (29)
was obtained by ligating the appropriate coding sequence (amino acids
241 to 374) downstream and in-frame with a sequence coding for the DNA binding domain of the yeast transcription factor Gal4 (amino acids 1 to
147) in the vector pFA-CMV (Stratagene). The PKA mutant of AD3
construct (serine residue 347 changed to alanine in PKA consensus phosphorylation site) was created by Quick Change site-directed mutagenesis kit (Stratagene). These constructs were verified by sequencing using the Sequenase kit (Amersham Pharmacia Biotech) and
tested for their ability to transactivate a luciferase reporter gene
driven by five copies of the Gal4 upstream activation sequence and the
adenovirus E1b minimal promoter in pFR-Luc (Stratagene).
-D-thiogalactoside. After 5 h of
induction, the cells were harvested. The histidine-tagged protein was
purified by His·Bind resin as recommended by the manufacturer (Novagen). The recombinant proteins also contained S-tag. The recombinant protein (2 µg) was incubated in a final volume of 20 µl
containing 20 mM Tris (pH 7.4), 10 mM
MgCl2, 50 µM ATP, 5 µCi of
[32P]ATP, and purified PKA catalytic subunit (Promega).
The reaction was carried out at 37 °C for 15 min and stopped by the
addition of SDS-PAGE loading buffer. The samples were separated by
SDS-PAGE and blotted onto polyvinylidene difluoride membrane. The
membrane was first exposed to autoradiography and then analyzed by
Western blot using horseradish peroxidase-conjugated anti-S-tag protein as recommended by the manufacturer (Novagen).
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-actin cDNA. That there was
no change in the levels of Cbfa1 RNA or protein in UMR cells under
either control conditions or after PTH treatment (Fig. 1, A
and B) suggests that PTH regulates Cbfa1
posttranslationally.
View larger version (28K):
[in a new window]
Fig. 1.
A, Western blot analysis of Cbfa1. Fifty
µg of total cellular lysate isolated from control and PTH-treated (2 h) UMR cells were used for Western blot analysis using Cbfa1 antibody
(anti-human AML-3). The arrow indicates a major band (~45
kDa) found in both control and PTH-treated lysates. B,
Northern blot analysis of Cbfa1 mRNA abundance. One µg of
poly(A)+ mRNA isolated from control and PTH-treated (2 h) UMR cells was used for Northern blot analysis using a mouse-specific
Osf2/Cbfa1 riboprobe. The filter was stripped and reprobed with
a -actin cDNA probe to verify an equal amount of RNA loading and
transfer.
148 C'ase-3) was transiently
transfected into UMR cells. The cells were then pretreated with PD98059
(ERK pathway inhibitor) or GF109203X (PKC inhibitor) for 20 min, then treated with or without PTH (10
8 M) for
24 h. Lysates were then assayed for CAT activity (Fig. 2). The CAT activity was not affected by
these inhibitors, suggesting that the ERK and PKC, signaling pathways
are not involved in the PTH stimulation of collagenase-3 promoter
activity. When we used H-89 (PKA inhibitor), PTH-induced collagenase-3
promoter activity was inhibited (Fig.
3A). In addition, we have also
transfected PKI, the heat-stable inhibitor of PKA (Fig. 3B),
and the result clearly indicated that PTH stimulation of collagenase-3
promoter activity was abrogated by PKA inhibition. Hence, PTH-induced
collagenase-3 promoter activity is mediated by the PKA pathway.
View larger version (19K):
[in a new window]
Fig. 2.
Effect of ERK pathway or PKC inhibitors on
collagenase-3 promoter activity. The collagenase-3 promoter
construct ( 148 C'ase-3) was transiently transfected into UMR cells
and then treated with either PD98059 (ERK inhibitor) (A) or
GF109203X (PKC inhibitor) (B) for 20 min. This was followed
by incubation in control or PTH (108
M)-containing media for 24 h and assessment of CAT
activity. Background was defined as the activity of the promoterless,
enhancerless vector, pSV0CAT. Data represent the mean ± S.E. of
three wells.
View larger version (18K):
[in a new window]
Fig. 3.
Effect of inhibition of PKA on collagenase-3
promoter activity. A, the collagenase-3 promoter
construct ( 148 C'ase-3) was transiently transfected into UMR 106-01 cells, then treated with H-89 (PKA inhibitor) for 20 min followed by
control or PTH (108 M)-containing media for
24 h and assayed for CAT activity. B, the collagenase-3
promoter construct was transiently cotransfected into UMR 106-01 with
the indicated amounts of PKI, the heat-stable inhibitor of PKA
catalytic subunit, followed by control or PTH (108
M)-containing media for 24 h, and assayed for CAT
activity. Background was defined as the activity of the promoterless,
enhancerless vector, pSV0CAT. Data represent the mean ± S.E. of
three wells. Cm, chloramphenicol.
To distinguish the primary effect of PKA on c-fos promoter
activity from Cbfa1 transactivation, we utilized the heterologous Gal4
transcription factor system. A region of 135 amino acids within the
proline-, serine-, threonine-rich domain of Cbfa1, C-terminal to the
DNA binding domain (runt), called AD3, is required for
transactivation (29). Unlike AD1 and AD2, AD3 can function in a
heterologous system (29), and it contains consensus phosphorylation sites for PKA, PKC, casein kinase II, and ERK. Since the collagenase-3 promoter activity was ablated by inhibition of PKA, and not by PKC or
ERK inhibitors, we concentrated on the role of PKA activity on Cbfa1
transactivation. To examine the role of PKA in the transactivation of
Cbfa1, we generated Gal4 AD3 and the PKA site mutant Gal4 AD3 expression vectors. These constructs were transiently cotransfected into UMR cells with an expression construct coding for the PKA catalytic subunit as well as a luciferase reporter gene containing Gal4
binding sites (Fig. 4A). The
wild type AD3 construct demonstrated enhanced luciferase activity when
the vector for PKA was cotransfected, indicating that the PKA site in
the wild type AD3 construct is functional and mediates the AD3
transactivation. To determine the PTH-stimulated AD3 transactivation,
the wild type AD3 construct was transiently transfected into UMR cells
and then treated with control or PTH (108
M)-containing media (Fig. 4B). The AD3 of Cbfa1
had enhanced luciferase activity when the cells were treated with PTH,
whereas cotransfection with PKI, a dominant inhibitor of PKA, inhibited the PTH-induced activity. These results show that the transactivation of AD3 by PTH is mediated by the PKA signaling pathway. When the PKA
site mutant AD3 construct was transfected into UMR cells, no
PTH-induced luciferase activity was detected (Fig. 4C). This further supports the previous data that the PTH-induced transactivation of AD3 in Cbfa1 is mediated by PKA.
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To determine whether AD3 can be phosphorylated by PKA, the wild type
and PKA site mutant AD3 of Cbfa1 were cloned into vectors enabling
expression in E. coli as His- and S- tag fusion proteins. The purified AD3 proteins were subjected to in vitro
phosphorylation by PKA (Fig.
5A). The recombinant wild type
AD3 was phosphorylated by PKA, whereas mutation of the PKA site
abolished phosphorylation of AD3 by PKA. Autophosphorylation of PKA was
also observed by incubating PKA in phosphorylation buffer without
substrate (AD3). The purified recombinant AD3 proteins contain an S
tag. Western blot analysis with horseradish peroxidase-conjugated
anti-S-tag protein was used to verify that equal amounts of the wild
type and PKA mutant AD3 were present in the phosphorylation reaction (Fig. 5B). These results indicated that PKA can
phosphorylate the wild type AD3, and this is mediated by the identified
PKA site. Thus, the PKA phosphorylation site within AD3 is the likely site for PKA-mediated transactivation of Cbfa1.
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Previous studies from our laboratory showed enhanced binding of c-Fos and c-Jun proteins at the AP-1 site upon treatment with PTH. However, there was no significant change in the level of Cbfa1 binding to the RD site (15). By Western and Northern blot analyses, we show here that there is no change in the levels of Cbfa1 RNA or protein in UMR cells under either control condition or after PTH treatment (Fig. 1, A and B). The presence of three Cbfa1 transcripts in the UMR cells (Fig. 1B) could be due to the difference in their N-terminal sequences or the alternative splicing, which leads to selective loss of exons. Several isoforms of Cbfa1 have been identified by differential promoter usage or differential splicing (30). Merriman et al. (19) report the presence of two transcripts in ROS 17/2.8. By Western blot, we found a minor protein of ~35 kDa in addition to the predominant ~45 kDa band showing that Cbfa1 antibody (anti-human AML-3 raised against a C-terminal peptide) will cross-react with other Cbfa1 isomeric forms having different N-terminal amino acid sequences (30). Recently, investigation of the functional differences of the three Cbfa1 isoforms (26, 33, 34) has shown that all three isoforms are involved in the stimulatory action of osteoblast differentiation, but they exert different functions in the process of osteoblast differentiation (35). In our studies, no change in the levels of Cbfa1 RNA or protein in UMR cells either in control or PTH-treated cells suggests posttranslational modification of Cbfa1 in response to PTH. It cannot be ruled out that different activities of isomeric forms of transcription factors generated by alternative splicing may result from different interaction with cofactors (36). It is also possible that PTH regulates Groucho/TLE2, a repressor protein (29), resulting in the alteration of Cbfa1 transactivation. The posttranscriptional regulation of Cbfa1 for TRI promoter activity in glucocorticoid-treated bone cells has also been proposed (37) but does not appear to be a factor here since there are no changes in mRNA or protein for Cbfa1.
PTH binds to the PTH/PTH-related protein receptor on osteoblastic cells
and generates multiple second messengers including cAMP (which
activates PKA), diacylglycerol (which activates PKC), inositol
trisphosphate, and increased levels of intracellular calcium (38, 39).
The transient transfection experiments of collagenase-3 promoter with
PKA, PKC, and ERK inhibitors in UMR cells clearly indicate that PTH
stimulation of collagenase-3 promoter activity is completely abrogated
by PKA inhibition and not by inhibition of the other pathways. Other
studies in our laboratory have shown that the ERK pathway inhibitor
PD98059 inhibits ERKs (ERK1 and ERK2) at 50 µM and the
PKC pathway inhibitor GF109203X inhibits PKC at 1 µM in
UMR cells (data not shown). If PTH-induced collagenase-3 promoter
activity is regulated through either the ERK or the PKC signaling
pathway, then those inhibitors used at the concentrations in these
studies should have inhibited the PTH effect. This is not the case.
Many genes such as 1(I) collagen (40), plasminogen
activator inhibitor-1 (41), leukemia inhibitory factor (3),
sodium/proton exchanger NHE-1 isoform (42), and PTH/PTH-related protein
receptor (43, 44) are regulated by PTH through the PKA-mediated
pathway. PTH induction of prostaglandin G/H synthase-2 (5),
interleukin-6 (45, 46), and ICER (inducible cAMP early repressor) (47)
in osteoblastic cells are also mediated by the PKA pathway. By
individually expressing distinct, stably transfected cAMP-, serum-, and
phorbol ester-inducible luciferase genes, it has been shown that
PTH-induced cAMP/PKA signaling pathway plays a predominant role in UMR
cells (48).
Recently we also showed that PKA is the enzyme responsible for phosphorylating CREB in response to PTH and that PKA activity is required for PTH-induced c-fos expression (32). Since PTH-induced collagenase-3 promoter activity is mediated by the PKA signaling pathway (Fig. 3), the primary effect of PKA-mediated CREB phosphorylation was distinguished from Cbfa1 transactivation by using the yeast Gal4 transcription factor system. The transient transfection studies with the wild type and the PKA mutant Gal4 AD3 constructs indicate that the transactivation of AD3 in Cbfa1 is dependent on the presence of the PKA consensus phosphorylation site and PTH induces the transactivation of AD3 through the PKA site (Fig. 4). Thus, the PKA site in AD3 of Cbfa1 is necessary for PTH-induced collagenase-3 promoter activity. Since the assembly of transcriptional proteins has important implications for the accuracy and diversity of transcriptional regulation in vivo, it cannot be ruled out that all three activation domains of Cbfa1 may be required for full transcriptional activity in the context of native protein by directly or indirectly interacting with other transcription factors like the AP-1 factors to regulate PTH-induced collagenase-3 promoter activity.
It is well known that protein phosphorylation is one of the most important processes for cellular regulation and signal transduction in eukaryotic cells and phosphorylation of transcription factors often plays a key role in modulating DNA binding and/or functional activity. In vitro phosphorylation studies indicate that the wild type AD3 is a substrate for phosphorylation by PKA (Fig. 5), suggesting that PTH-induced transactivation of Cbfa1 is mediated by PKA phosphorylation of AD3. Taken together (Figs. 4 and 5), we propose that the mechanism by which PTH stimulates the rat collagenase-3 promoter is via phosphorylation and activation of Cbfa1 by PKA in addition to the enhanced level of c-Fos and c-Jun proteins. Since we did not observe increased binding of Cbfa1 to the RD site of collagenase-3 promoter in response to PTH by gel shift experiments (15), it is likely that the phosphorylated Cbfa1 may not have increased DNA binding activity. Similarly, it has been shown that the Cbfa2 phosphorylated in response to cytokines is unchanged in DNA binding activity (49). It is possible that the phosphorylation of Cbfa1 could lead to enhanced interaction with AP-1 factors either directly or indirectly for PTH-induced collagenase-3 promoter activity. In our laboratory, we have also used other osteoblastic cells to study the PTH effect on collagenase-3 expression. We have found that the effect of PTH on collagenase-3 mRNA expression in normal, differentiating osteoblasts and MC3T3 cells is only 2-3-fold (50).2 In ROS 17/2.8 cells, collagenase-3 is not induced in response to PTH, even though these cells are responsive to PTH in terms of production of cAMP.3 Hence, it may be difficult to dissect the signaling pathway for PTH-induced collagenase-3 promoter activity in other osteoblastic cells.
In summary, we have found that the PKA site of AD3 in Cbfa1 appears to
be physiologically important and involved in PTH action and could also
be involved in prostaglandin E2 action through the PKA
pathway. Mutation of this site could possibly impair PTH-induced bone
resorption, since this site is involved in PTH-induced collagenase-3 promoter activity.
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ACKNOWLEDGEMENTS |
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We thank Dr. Scott Hiebert for the human anti-Cbfa1 antibody and Dr. Gerard Karsenty for the mouse Cbfa1 (Osf2/Cbfa1) cDNA clone.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants DK47420 and DK48109 (to N. C. P) and a National Osteoporosis Foundation grant (to N. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of
Pharmacological and Physiological Science, St. Louis University School of Medicine, 1402 South Grand Blvd., St. Louis, MO 63104. Tel.: 314-577-8551; Fax: 314-577-8233; E mail: Partrinc{at}slu.edu.
2 R. C. D'Alonzo and N. C. Partridge, unpublished data.
3 N. Selvamurugan, R. J. Brown, and N. C. Partridge, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
PTH, parathyroid
hormone;
AP-1, activator protein-1;
RD, runt domain;
AML, acute myelogenous leukemia;
Osf2, osteoblast specific factor-2;
Cbfa1, core binding factor 1;
AD3, activation domain-3;
PKA, protein
kinase A;
PKC, protein kinase C;
ERK, extracellular signal-regulated
kinase;
CREB, cAMP-response element-binding protein;
CAT, chloramphenicol acetyl transferase;
PAGE, polyacrylamide gel
electrophoresis;
MOPS, 4-morpholinepropanesulfonic acid;
C'ase, collagenase.
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REFERENCES |
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