(Received for publication, October 20, 1995; and in revised form, January 23, 1996)
From the
Fibroblast growth factors (FGFs) are important regulators of calvarial osteoblast growth and differentiation. We have studied the regulation of the osteoblast-specific gene osteocalcin (OC) by FGF2 in phenotypically immature MC3T3-E1 calvarial osteoblastic cells. FGF2 markedly induces OC mRNA accumulation in MC3T3-E1 cells in the presence of forskolin (FSK). Similarly, OC promoter activity (luciferase reporter) is up-regulated 6-10-fold by FGF2/FSK or by FGF2/8-bromo cyclic AMP. Half-maximal induction of OC promoter activity occurs at 1 nM FGF2. By 5` deletion analysis and dinucleotide point mutations, we map one component of this FGF2/FSK response to a GCAGTCA motif in the region -144 to -138 relative to the OC transcription initiation site. The OC promoter region -154 to -90 confers FGF2/FSK responsiveness on the Rous sarcoma virus minimal promoter. By 3` and internal deletion analyses, the region between -90 to -99 is also found to be necessary for FGF2/FSK synergy (encodes a PuGGTCA motif previously identified as a component of FSK induction). A DNA binding activity that recognizes the region -148 to -125 of the rat OC promoter is induced in crude nuclear extracts from MC3T3-E1 cells treated with FGF2 or FGF2/FSK. This binding activity is sequence-specific and does not recognize the TCAGTCA DNA cognate of AP1. Members of the ATF, Fos, and Jun family are not immunologically detected in this inducible DNA binding activity. However, transient co-expression of ATF3 but not ATF2 selectively attenuates the FGF2 component of induction. Thus, a novel FGF2-regulated DNA-protein interaction in the OC promoter participates in the transcriptional control of OC expression by FGF and cyclic AMP in MC3T3-E1 calvarial osteoblasts.
FGF ()family members control osteoblast gene
expression in a biphasic fashion, dependent upon the stage of
osteoblast
maturation(1, 2, 3, 4, 5, 6) .
Dominant mutations in FGF receptor -1 and FGF receptor -2
give rise to abnormal skeletal phenotypes, including craniosynostosis
characterized by accelerated intramembranous calvarial bone formation
in the cranial
sutures(7, 8, 9, 10) . However,
virtually nothing is known of the transcriptional mechanisms whereby
FGF regulates expression of the osteoblast phenotype. Using fetal calf
calvarial bone cells, Gospodarowicz and co-workers (1) demonstrated that FGF2 (basic FGF) stimulates osteoblast
proliferation and the production of osteocalcin, a bone-specific
protein(11, 12, 13) . Recent in vivo studies using rats have shown that intravenous FGF2 stimulates
bone formation and mineralization(3, 4) . Similarly,
FGF2 potentiates expression of the osteoblast phenotype (including
osteocalcin production) in a subset of immature, preosteoblastic
stromal cells known as colony-forming cells(5, 6) ; by
contrast, phenotypically more mature osteoblast cluster-forming cells
do not respond to FGF2 in this way. Studies using phenotypically
mature, ROS 17/2.8 osteosarcoma cells (2, 14) reveal
that FGF can act to globally suppress features of the differentiated
osteoblast via a pertussis toxin-sensitive G protein(2) .
Whether FGF2 signaling is influenced by sarcomatous transformation has
not been systematically examined; however, a novel oncogenic guanine
nucleotide exchange factor, ost, has been isolated from ROS
17/2.8, suggesting that G protein signaling may be altered in this
cellular background(15) .
MC3T3-E1 cells are a
phenotypically immature, mouse calvarial osteoblast cell line cloned
from spontaneously immortalized calvarial cells selected by the 3T3
passaging protocol(16) . MC3T3-E1 cultures sequentially
elaborate osteoblast phenotypic
markers(16, 17, 18) , closely mimicking
results obtained with cultures of primary calvarial osteoblasts
(reviewed in (19) ). The osteoblast-specific phenotypic marker,
osteocalcin (OC), is initially expressed at low levels and then
up-regulated with time in culture and ascorbic
acid(17, 18) . MC3T3-E1 cells respond to FGF2 (20) and like normal calvarial
osteoblasts(7, 8, 9) express mRNAs for FGF
receptors -1 and -2. ()Previously, we have shown
that the osteoblast-specific rat OC promoter is active in MC3T3-E1
cells (21, 22, 23) . Thus, the MC3T3-E1 cell
culture model provides a readily manipulable system for studying how
hormonal signals, including FGF2, interact with the calvarial
osteoblast transcriptional machinery assembled by the OC promoter.
In this work, we show that FGF2 up-regulates OC mRNA accumulation and OC promoter activity in phenotypically immature MC3T3-E1 cultures in the presence of cyclic AMP. We map one component of this novel FGF2-dependent response to a GCAGTCA motif at nucleotides -144 to -138 in the osteoblast-specific (19, 21, 24) proximal promoter region of rat OC. A DNA binding activity that recognizes this region of the rat OC promoter is up-regulated in crude nuclear extracts from MC3T3-E1 cells treated with FGF2 or FGF2/FSK. By internal deletion analysis in the context of the homologous OC promoter and 3` deletion analysis in the context of a heterologous minimal promoter, we show that a second region, -90 to -99, previously defined as a component of cyclic AMP responsiveness(23) , is necessary for FGF2/FSK induction. Intriguingly, FGF2 induction of the OC promoter is selectively inhibited by ATF3, a transcriptional repressor (25) involved in FGF-dependent up-regulation of proenkephalin gene expression(26) . Thus, a novel FGF2-regulated DNA-protein interaction in the OC promoter participates in the transcriptional control of OC expression in MC3T3-E1 calvarial osteoblasts.
Figure 1: FGF2 and FSK synergistically up-regulate OC mRNA accumulation in phenotypically immature MC3T3-E1 calvarial osteoblasts. MC3T3-E1 cells were cultured as described under ``Experimental Procedures,'' treated for 18 h with vehicles, 10 µM FSK, 6 nM FGF2, or both, and total RNA was harvested(29) . Messenger RNA levels for OC, COL1A1, and GAPD were assessed using a semiquantitative reverse transcription-PCR technique as previously detailed (28) and as outlined in the text. Note that the combination of FGF2 and FSK up-regulates OC mRNA levels but not GAPD levels. Note also that this regulatory effect and interaction is selective, because suppression of COL1A1 by FGF2 (20) occurs either in the presence (lane 4) or the absence (lane 3) of FSK. CON, control.
Figure 2: FGF2 and FSK synergistically up-regulate OC promoter activity. MC3T3-E1 cells were transfected with 1050 OCLUC and 795 BSPLUC as described under ``Experimental Procedures.'' After a 2-day recovery, cells were treated for 18 h with vehicles, 10 µM FSK, 0.3 nM FGF2, or both as indicated. Extracts were subsequently prepared and analyzed for luciferase activity as described previously(21, 22, 23) . Note that the combination of FGF2 and FSK synergistically up-regulates the rat OC promoter. Note also that this effect is promoter-specific, because the BSP promoter is suppressed not synergistically activated. The FSK effect can be mimicked by 0.2 mM 8-bromo cyclic AMP ( (23) and not shown).
Figure 3:
FGF2 induction of the OC promoter in the
presence of 10 µM FSK. Note that half-maximal induction of
OC promoter activity by FGF2 occurs at 1 nM. FSK by
itself induces promoter activity 2.5-fold via an element located
between -121 and
-74(23) .
Figure 4: The synergistic induction of basal OC promoter activity is dependent upon a motif located between -154 and -138 relative to transcription initiation. A series of rat OC promoter 5` deletion constructs with luciferase reporter were transfected into MC3T3-E1 cells and examined for induction in the presence of FSK (10 µM) and FGF2 (1.5 nM) as described in the legend to Fig. 2. Note that synergistic induction is markedly attenuated upon deletion of the region -154 to -138.
Figure 5:
An intact GCAGTCA motif at -144 to
-138 in the OC promoter is necessary for synergistic induction by
FGF2/FSK. A series of dinucleotide substitutions were made within
context of the homologous -154 to 32 OC promoter fragment (154
OCLUC). Note that substitutions upstream (154 OCMUT#1-#2) or downstream
(154 OCMUT#6-#7) of the region -144 to -138 had little
effect on FGF2/FSK induction. By contrast, note also that the GCAGTCA
to AAAGTCA alteration seen in 154OCMUT#4 decreased FGF2/FSK induction
by 50%. Furthermore, the GCAGTCA to GCATTTA substitution in 154
OCMUT#5 completely abrogates the FGF2/FSK synergistic induction without
affecting the FSK response, consistent with the results obtained from
the 5` deletion analyses (see Fig. 4). Introduction of the
TTAGTCA motif for AP1 at this position (154 OCMUT#3) results in a
variant partially inducible by FGF2 alone (2-fold) and subsequently
more responsive FGF2/FSK. See text for
details.
Figure 11: Regulatory elements in the proximal rat OC promoter. Numbering is relative to the start site of transcription. Hex A and Hex B refer to the hexamer motifs (TGACCPy on top strand, PuGGTCA on bottom strand) of the cyclic AMP response region(23) . See text for details and references.
Figure 6:
The
OC promoter fragment -154 to -90 can confer FGF2/FSK
responsiveness to the RSV heterologous minimal promoter fragment. A, rat OC promoter fragments -154 to -74,
-199 to -74, -199 to -112, -154 to
-90, and -154 to -100 were placed in native
orientation upstream of the inactive ( (21) and data not shown)
RSVLUC minimal promoter fragment -51 to 35. These constructs were
transfected into MC3T3-E1 cells, and cultures were subsequently treated
with vehicles, 10 µM FSK, 6 nM FGF2, or both as
described in the text. As shown, the OC promoter fragment -154 to
-74 confers FGF2/FSK responsiveness to the RSVLUC minimal
promoter. Deletion of the OC promoter region -74 to -111 in
the context of a larger OC promoter fragment completely abrogates the
FGF2/FSK response (-199 to -74 versus -199
to -112). B, the smaller OC fragment -154 to
-90 also confers FGF2/FSK responsiveness to the RSVLUC minimal
promoter. Deletion of the 10-base pair region between -90 and
-99, inclusive, completely abrogates the FGF2/FSK response
(-154 to -90 versus -154 to -100).
Internal deletion of the overlapping region -112 to -93 in
the homologous promoter context as in 154 (-112 to
-93) OCLUC also abrogates FGF2/FSK induction. See Fig. 11for OC promoter sequence.
Figure 7: ATF3 attenuates FGF2/FSK but not FSK induction of the OC promoter. The OC promoter construct 154 OCLUC (5.5 µg/ml) was co-transfected with empty expression vector (CONTROL), pCG-ATF2, or pCG-ATF3 (1.8 µg/ml) as indicated. After a 2-day recovery period, cells were treated for 18 h with vehicles, 10 µM FSK, 6 nM FGF2, or FGF2/FSK and subsequently analyzed for luciferase activity. Note that ATF3 attenuates FGF2/FSK induction but not FSK induction of the OC promoter. Note also that ATF2 did not inhibit promoter induction.
Figure 8: FGF2 and FGF2/FSK up-regulate a DNA binding activity in nuclear extracts that recognize the rat OC promoter region -146 to -125. MC3T3-E1 cells were cultured as described under ``Experimental Procedures'' and treated with vehicles, 10 µM FSK, 6 nM FGF2, or both for 18 h. Crude nuclear extracts were prepared and analyzed for DNA binding proteins by gel shift assays as detailed in the text. Lanes 1 and 6, no nuclear extract (control, CON). Lanes 2-5 and 7-10, 5 µg of nuclear extract/reaction. In the absence of nuclear extract, no DNA-protein complexes are observed (lanes 1 and 6). Note that FGF2 (lane 3) and FGF2/FSK (lane 4) markedly increase binding to the rat OC promoter region -146 to -125 (FRE oligo); a smaller increase is noted with FSK treatment alone (lane 2). AP1 binding activity is significantly up-regulated by FSK, FGF2, or the combination (lanes 7-10). Note, however, that the FRE DNA-protein complex and the AP1 DNA-protein complex do not co-migrate (arrows).
Figure 9:
The FGF2-inducible DNA binding activity
recognizing the rat OC promoter fragment -146 to -125 does
not recognize the AP1 or thyroid hormone response element palindrome
DNA cognates. The specificity of the FGF2 inducible DNA-protein
interaction observed in Fig. 8was examined by binding
competition with excess unlabeled DNA cognates as described
previously(21, 22, 23) . A, lane
1, no nuclear extract; lanes 2-6, 6 µg of
nuclear extract/binding reaction from FGF2-treated MC3T3-E1 cells. In
the absence of nuclear extract, no gel shift is observed (lane
1). The addition of 30-fold molar excess of unlabeled FRE OC
promoter oligo inhibited binding to the homologous labeled FRE fragment (lane 3). However, 30-fold (lane 5) and 90-fold (lane 6) molar excess of unlabeled AP1 cognate did not compete
for binding the FGF2 inducible factor (Fig. 8) to the OC FRE
promoter fragment. The radiolabeled FRE oligo concentration was 1.5
10
M. B, lane 1,
no nuclear extract; lanes 2-10, 6 µg of nuclear
extract per tube from MC3T3-E1 cells treated with FGF2. Note that
50-fold molar excess of thyroid hormone response element palindrome
(with sequence AGGTCA) could not compete for binding (lane 7).
By contrast, 50-fold excess of the unlabeled rOC BOX (with sequence
GGGGTCA) weakly competes for binding (lane 10) to a lesser
degree than that observed with the homologous unlabeled FRE oligo (with
sequence GCAGTCA; lane 4).
Figure 10: The DNA-protein complex assembling on the OC promoter region -146 to -125 does not contain common Fos, Jun, or ATF family members. Nuclear extracts from MC3T3-E1 cells were preincubated with antibodies to Fos, Jun, or ATF family members as described under ``Experimental Procedures'' before proceeding to gel shift assays. Lane 1, no nuclear extract. Lanes 2- 10, 6 µg/reaction of nuclear extract from MC3T3-E1 cells treated for 18 h with FGF2. A, DNA-protein complexes assembling on the OC promoter fragment -146 to -124 (FRE). Note that none of the antibody treatments indicated disrupted or supershifted the FRE oligo DNA-protein complex. B, DNA-protein complexes assembling on the AP1 DNA cognate. Note that the anti-Fos (lane 3) and anti-Jun (lane 4) antibodies partially supershifted (lanes 3 and 4) and disrupted (lane 3) the DNA-protein complex binding to the AP1 element.
Previously, we identified a cyclic AMP response region (-121 to -74) in the proximal osteocalcin promoter ( (23) and Fig. 11). We now describe a novel element participating in a FGF2 transcriptional response, which cooperates with cyclic AMP to synergistically induce OC expression and which binds a DNA complex up-regulated in cells treated with FGF2 or FGF2/FSK. To our knowledge, this is the second promoter element shown to be synergistically activated by FGF2/FSK. Tan et al.(26) recently described a proenkephalin promoter element stimulated by FGF/FSK via ATF3 and c-Jun. However, the inducible MC3T3-E1 protein complex assembling on the rat OC promoter does not contain ATF3 or c-Jun. Moreover, co-expression of ATF3 inhibits FGF2/FSK induction of the OC promoter, consistent with the recently defined role for ATF3 as a transcriptional repressor(25) . Thus, the character of the OC promoter FGF2/FSK response differs from that of the proenkephalin promoter(26) .
The sequence of the
FRE and the observation that ATF3 inhibits induction introduces the
notion that a known or novel member of the leucine zipper family (32, 33) may be regulating the OC promoter via this
element. The similarity of the GCAGTCA FRE motif to the TPyAGTCA AP1
motif prompted us to carefully examine whether common members of the
Fos and Jun family were present in the DNA-protein complex assembling
on the OC FRE. Alteration of the OC promoter FRE to fit the AP1
consensus does enhance FGF2/FSK induction but also results in basal
induction by FGF2 in the absence of FSK, a response not noted with the
native OC promoter. Three sets of data strongly suggest that the
FGF-inducible DNA-protein interaction at the native OC FRE does not
contain AP1 factors: (i) The complex assembling on the OC FRE does not
recognize the classic AP1 cognate. (ii) The complex assembling on the
OC FRE does not supershift with -Fos or
-Jun antibodies,
whereas the complex assembling on the AP1 element does supershift with
these same reagents. (iii) Tetradecanoyl phorbol acetate, which
activates AP1 via protein kinase C, cannot replace FGF2 in the
synergistic interaction with FSK. (
)Moreover, Stein and Lian (19) have shown that AP1 activity suppresses the OC promoter
during osteoblast proliferation(19) ; this also suggests that
AP1 is unlikely to be directly participating in OC promoter induction
via the FRE.
The capacity of FGF2 and FSK to synergistically
activate OC expression and promoter activity in MC3T3-E1 cells is
intriguing. In fibroblasts (33) and osteosarcoma
cells(34) , cyclic AMP inhibits mitogen-activated protein
kinase activation at the level of Raf-1 interaction with Ras (33) . FGF2 does activate mitogen-activated protein
kinase(34, 35) . However, it was recently shown that
FGF2 elaborates unique signals that suppress the myogenic
differentiation program of MM14 cells; independent activation of the
mitogen-activated protein kinase cascade by platelet-derived growth
factor is not sufficient to suppress MM14 skeletal muscle gene
expression(35) . Similarly, preliminary studies show that
epidermal growth factor does not synergize with FSK to up-regulate OC
in MC3T3-E1 osteoblasts. Taken together, these observations
introduce the notion that FGF receptors may initiate intracellular
signal cascades in calvarial osteoblasts that are overlapping yet
distinct from those of other mitogen receptor tyrosine kinases, a
notion to be directly examined in future studies of MC3T3-E1 gene
regulation.
The precise mechanisms whereby FGF2 and FSK synergize to up-regulate OC promoter activity remain to be detailed. Possibilities include independent regulation of protein DNA interactions at the the FRE and cyclic AMP response region (23) or regulation of obligate protein-protein interactions between factors bound to these elements. Consistent with both mechanisms, FGF2/FSK induction requires the two elements encompassed by the OC promoter fragment -154 to -90. By both 5` deletion analysis and point mutagenesis in the context of the native OC promoter, we directly demonstrate that the region -144 to -138 is necessary for the FGF2/FSK response. However, it is not solely sufficient for this response; three tandem copies of the sequence CTGCAGTCAC (-146 to -137) cannot reconstitute the response when placed upstream of the RSV minimal promoter, even though it readily competes for nuclear factor binding to the FRE oligo. We and others have mapped important basal regulatory elements both upstream and downstream of the OC FGF response element (19-24, 38-42; Fig. 11). Furthermore, Ducy and Karsenty (24) have functionally defined an element contiguous to the FRE that plays an important role in the cell type specificity of the proximal mouse and rat OC promoters(19, 21, 24, 42) . However, the FGF-responsive element is distinct from this element, because mutations that destroy factor binding to this element (24, 42) do not disrupt FGF2/FSK synergy. The interplay between tissue-specific and stimulus-specific OC promoter elements remains to be detailed. Regardless of the mechanism, in MC3T3-E1 cells the OC promoter is functionally acting as a coincidence detector, responding to bipartite inductive signals elaborated by FGF2 and FSK. The precise nature of this response will require identification and characterization of the proteinaceous factors binding hormonally responsive regions of the OC promoter and should provide a more detailed understanding of how FGF-elaborated signals impact osteogenesis in development and disease(5, 7, 8, 9) .
Finally, Schedlich et al.(43) recently described a FGF response element in the human OC promoter. The element they describe differs from the rat OC FRE in several aspects: (i) The human OC element is inducible by FGF2 alone. (ii) The human OC element resembles an NF-1 DNA cognate. (iii) The human OC element maps to a distal upstream promoter fragment, near the vitamin D response element. Moreover, their studies (43) were carried out in phenotypically mature, transformed ROS17/2.8 osteosarcoma cells, where the endogenous OC mRNA expression is down-regulated by FGF(2) . Future studies will be necessary to systematically examine the influences of sarcomatous cellular backgrounds and the different stages of osteoblast maturation upon the regulation of these elements by FGF2.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) J04500[GenBank].