 |
INTRODUCTION |
Elaboration of the vertebrate skeleton occurs via two overlapping,
yet distinct developmental pathways. Intramembranous ossification, the
primary pathway for flat bone development, relies upon the direct
differentiation of condensed mesenchyme into osteoblasts. These
osteoblasts then secrete various matrix components until they are
encapsulated by calcified bone, thus linking the rate of
intramembranous bone growth to the rate of osteoblast differentiation. Alternatively, endochondral ossification, the primary pathway for
formation of the axial and appendicular skeleton, differs from
intramembranous ossification in that condensed mesenchymal cells
(chondrocytes) elaborate a complex cartilaginous template as they
progress through a series of developmental stages at the epiphyseal
growth plate. The epiphyseal growth plate is organized into distinct
cellular compartments: the resting zone, which serves as a renewable
source of chondrocytes; the proliferative zone, where rapid cell
division results in stacked columns of chondrocytes; and the
hypertrophic zone, where the cells terminally differentiate, hypertrophy, and secrete a specialized matrix. After the encapsulated hypertrophic chondrocytes mature, the associated extracellular matrix
is rapidly invaded by blood vessels and bone-forming osteoblasts, which
synthesize trabecular bone. Thus, the overall rate of longitudinal bone
growth is regulated by the progression of chondrocytes through these distinct developmental stages within the epiphyseal growth plate.
In vitro and in vivo model systems have been
utilized to study both the commitment and subsequent differentiation of
chondrocytes (1-5). These studies demonstrate that many physiological
effectors are capable of modulating chondrocyte differentiation
in vitro and that mutations or alterations in gene
expression can result in skeletal defects in vivo. Recent
genetic evidence has linked eight different craniosynostosis and
chondrodysplasia syndromes to mutations in three of the
FGFRs1 (6, 7). These
autosomal dominant mutations affect both intramembranous and
endochondral ossification, suggesting that FGF signaling is an
essential regulator of skeletal development.
Mutations in the fgfr3 gene have been linked to several
skeletal dysplasias in humans, including achondroplasia (8, 9), thanatophoric dysplasia types I and II (10, 11), and hypochondroplasia (12). Achondroplasia is the most common genetic form of dwarfism in
humans and results from a mutation in the transmembrane domain (G380R)
of FGFR3, while thanatophoric dysplasia is the most common neonatal
lethal skeletal dysplasia in humans and results from any of three
independent point mutations in the fgfr3 gene. Clinically, all of these mutations result in a characteristic disruption of growth
plate architecture and disproportionate shortening of the proximal
limbs. Biochemically, these mutations activate FGFR3 signaling (13,
14). In contrast, inactivation of FGFR3 signaling in mice results in an
increase in the size of the hypertrophic zone, as well as a coincident
increase in bone length (15, 16). Such complementary phenotypes suggest
that FGFR3 signaling is an essential component of the hierarchy of
regulatory mechanisms that regulate endochondral bone growth.
Recent studies have demonstrated that PTH/PTHrP receptor signaling,
like FGFR3 signaling, regulates the process of endochondral bone growth
(17-20). Inactivation of either PTHrP or the PTH/PTHrP receptor in
mice results in the premature ossification of proliferating chondrocytes as well as a marked decrease in the size of the
proliferative zone (17, 18), a phenotype resembling that seen with
constitutive activation of FGFR3 signaling. The hypothesis that PTHrP
signaling regulates the proliferation of chondrocytes by modulating
FGFR3 activity provides an explanation for the similarity in these
phenotypes. Such control may involve transcriptional, translational,
and/or post-translational mechanisms. To this end, we have examined
whether PTH/PTHrP receptor signaling regulates the transcriptional
activity of the fgfr3 gene.
To examine the mechanisms governing expression of the fgfr3
gene in chondrocytes, an "enhancer" element capable of
recapitulating the chondrocytic expression pattern of fgfr3 in
vitro was identified and shown to be suppressed by PTH/PTHrP
receptor signaling. Furthermore, activation of protein kinase A (PKA)
signaling, either through the elevation of cAMP levels or through the
overexpression of the catalytic subunit of PKA, also suppressed the
transcriptional activity of the enhancer element and decreased the
steady state levels of the endogenous fgfr3 gene. These
results suggest a model whereby the attenuation of PKA
signaling, by effectors such as the PTH/PTHrP receptor,
serves to regulate endochondral ossification by modulating the
chondrocytic expression of fgfr3.
Electrophoretic mobility shift assays (EMSAs) were used to identify a
single hexameric "orphan" receptor half-site in the enhancer
element that interacts with nuclear protein(s). Elevation of cAMP
levels failed to disrupt this DNA-protein complex; however, mutations
in this motif that inhibit nuclear protein(s)/DNA interactions do
dramatically impair chondrocytic transcriptional activity. Nevertheless, these same mutations did not prevent the cAMP-mediated suppression of transcriptional activity, thus suggesting that there are
additional DNA/protein interactions within the enhancer element that
are required for the cAMP-dependent regulation of fgfr3 expression.
| |
EXPERIMENTAL PROCEDURES |
Plasmid Construction--
p(
2951/27)FR3-luc,
p(2311/27)FR3-luc, p(1537/27)FR3-luc, p(220/27)FR3-luc,
p(175/27)FR3-luc, p(126/27)FR3-luc, and p(79/27)FR3-luc
were constructed as described previously (21). To generate pCSR-luc,
the appropriate fragment was amplified from p(2311/27)FR3-luc using
the primers DM62 (5'-TGT ATC TTA TGG TAC TGT AAC TG-3') and DM144
(5'-GTC GAC CCA TGG GCC CAA CAG ACC-3'). The amplified product was
sequenced, digested with SalI and XhoI, and
concatamerized through self-ligation with T4 DNA ligase. A three-copy,
head-to-tail array was subcloned into pRSV-luc (22) digested with
XhoI. Orientation was confirmed by restriction endonuclease
analysis. Additional deletion constructs were generated using the same
protocol and the following synthetic oligonucleotides: pCSRa-luc, DM62,
and DM159 (5'-GTC GAC TTC CTG TAT TGA GCC GAC TTC C-3'); pCSRb-luc,
DM144, and DM160 (5'-CTC GAG GGA AGT CGG CTC AAT ACA GG-3'); pCSRc-luc,
DM62, and DM162 (5'-GTC GAC CTC AGT CTA GTC TCT GCC TCT CC-3');
pCSRd-luc, DM161 (5'-CTC GAG GGA GAG GCA GAG ACT AGA CTG AG-3') and
DM159; pCSRe-luc, DM163 (5'-CTC GAG ATG AAC AAC TCT AAA TCC-3'), and
DM164 (5'-GTC GAC AGA AGT CTG AGG TCA AGT TGG-3'); pCSRf-luc, DM62, and
DM175 (5'-GGG AGT GTC GAC TCC TCC CCA CGT CTC CTG-3'); pCSRg-luc, DM62,
and DM176 (5'-GGG AGT GTC GAC GGA TTT AGA GTT GTT CAT-3'); pCSRh-luc,
DM62, and DM177 (5'-GGG AGT GTC GAC CGT TAT GCT TGC AAG ACT-3'). A
trinucleotide substitution in the 5'-PuGGTCA-3' sequence motif of CSRh
was generated by polymerase chain reaction. The amplified product
(mCSRh) was sequenced, and a three-copy array was cloned into pRSV-luc
as described above.
To generate pCRE-luc, CREB-A (5'-GGT TGC CTG ACG TCA GAG A-3') and
CREB-B (5'-CCT CTC TGA CGT CAG GCA A-3') synthetic oligonucleotides (Life Technologies, Inc.) were 5'-phosphorylated with T4 kinase and
annealed. The annealed oligonucleotides were concatamerized by ligation
with T4 ligase, blunted with Klenow and deoxynucleotide triphosphates,
and then subcloned into the SmaI site of RSV-luc. Clones
were sequenced to verify insert identity and orientation. pCRE-luc (TL
IV 179.22) contains three copies of CREB-A in a head-to-tail array
upstream of the RSV minimal promoter.
Expression Plasmids and Other DNA Reagents--
The rat FGFR3
RNase protection probe was generated by amplifying rat cDNA with
the following primers: DO80, 5'-GTC ATG GAA AGT GTG G-3'; and DO113,
5'-GCT CCG ACA CAT TGG-3'. The resulting product was cloned into pGEM-T
(Promega Corp.), and both the identity and orientation of the insert
was confirmed through sequencing. The plasmid pTRI-
-Actin-125-Rat
was purchased from Ambion, Inc. The expression plasmid for the
catalytic subunit of protein kinase A (MT-C
) was obtained from S. McKnight (23), while empty vector control (pEV142) was provided by R. Palmiter (24). Expression plasmids for wild-type (HKrk) (25) and
mutated versions (HKrk-H223R (25) and HKrk-T410P (25)) of the human
PTH/PTHrP receptor were provided by H. Jüppner.
Cell Culture and Transfection--
CFK2 (26) and RCJ (27) (clone
3.1C5.18) cell lines were obtained from J. Henderson and J. Aubin,
respectively. Cell lines were maintained subconfluent in Dulbecco's
modified Eagle's medium (Life Technologies) supplemented with 10%
fetal calf serum (Sigma), 2 mM L-glutamine,
penicillin (100 units/ml), and streptomycin (100 µg/ml). All cells
were transfected in triplicate using a modified calcium phosphate
precipitant (28). The day prior to transfection, cells were plated at a
density of 8 × 104 cells/well in a 12-well plate
(Corning). Four hours prior to transfection, cells were provided with
0.8 ml of fresh media. Five micrograms of each DNA construct
(double-banded on a CsCl2 gradient) and 0.5 µg of
pCScyto-gal (29, 30) were diluted to a final volume of 20 µl with
distilled, deionized water. To that was added 250 µl of 0.272 M CaCl2 and 270 µl of 2× BBS (50 mM
N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic
acid (BES), 280 mM NaCl, 1.5 mM
Na2HPO4). The DNA mixture was then allowed to precipitate at room temperature for 15 min, and 80 µl of the
precipitant was then added directly to the cells. Cells were incubated
(5% CO2, 37 °C) with the precipitate for 20 h, at
which point they were then washed with Dulbecco's modified Eagle's
medium and returned to fresh media. When cells were exposed to various
pharmacological agents, cells were serum-starved (0.5% serum/Ham's
F-12 medium) for 12 h prior to treatment. Sixty hours
post-transfection, cells were washed with 1 ml of 1× PBS and
subsequently lysed in the well with 250 µl of luciferase lysis buffer
(25 mM Tris·HCl (pH 7.8), 2 mM dithiothreitol
(DTT), 2 mM
trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid , 10% glycerol, 1% Triton X-100). Insoluble materials were removed from all lysates by centrifugation at 14,000 rpm for 4 min, and
50-100 µl of the cleared lysate was used to analyze luciferase reporter expression as described previously (31). -Galactosidase activity was determined with the Galacto-Light Plus system, as described by the manufacturer (Tropix, Inc.).
Forskolin (Sigma) was dissolved in Me2SO (Sigma) to
generate a 1000× stock. The stock solution was diluted to 100× with
Ham's F-12 prior to addition to cells. Synthetic human PTH-(1-34)
(Sigma) was resuspended in 0.01 M acetic acid and stored at
80 °C. A 2000× stock of PTH was diluted 1:20 with Ham's F-12
medium prior to the stimulation of PTH/PTHrP receptor-transfected cells.
RNase Protection Analysis--
Gene expression was determined by
RNase protection analysis. Briefly, total cellular RNA was purified
from a confluent 10-cm dish using the RNeasy kit (Qiagen, Inc.). Twenty
micrograms of RNA was lyophilized prior to resuspension in 30 µl of
hybridization buffer (40 mM PIPES (pH 6.4), 400 mM NaCl, 1 mM EDTA, 80% formamide), which
contained 0.5-5×105 cpm of the appropriate
[-32P]UTP (800 mCi/mmol; NEN Life Science Products)
transcribed probes. Samples were denatured at 95 °C for 5 min and
then incubated at 50 °C for 8 h. Unhybridized probe was removed
by adding 350 µl of RNase digestion buffer (10 mM
Tris·HCl (pH 7.5), 300 mM NaCl, 5 mM EDTA, 14 µg of RNase A, 248 units of RNase T1) and incubating the reaction for
30 min at 30 °C. Duplex RNA hybrids were then purified with RNAzol B
(Teltest, Inc.), dried under vacuum, and resuspended in 8 µl of RNA
loading buffer (1 mM EDTA, 0.1% bromphenol blue, 0.1%
xylene cyanole FF, 80% formamide). Samples were denatured for 5 min at
95 °C, chilled on ice, and resolved on a 5% denaturing polyacrylamide gel (Long Ranger; J. T. Baker). Gels were dried under vacuum and exposed to Kodak X-Omat film. When RNase protection assays were quantified, gels were analyzed with a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA) and quantified with ImageQuant software (version 3.3, Molecular Dynamics). The sizes for
the protected fragments are as follows: rat FGFR3, 291 nt; rat
-actin, 126 nt.
Nuclear Extracts and EMSAs--
Nuclear extracts were prepared
according to the protocol of Dignam et al. (32). Briefly, 10 10-cm or six 15-cm plates of confluent cells were washed three times
with 1× PBS and scraped into 15 ml of 1× PBS. The cells were
collected by centrifugation at 1600 rpm for 5 min at 4 °C. The
supernatant was discarded, and the cells were washed with 5 cell
volumes of 1× PBS and pelleted as described above. The supernatant was
discarded, and the pellet was resuspended in 5 ml of buffer A (10 mM HEPES (pH 7.9, at 4 °C), 1.5 mM
MgCl2, 10 mM KCl, 0.5 mM DTT). The
cell suspension was then incubated for 10 min at 4 °C, transferred
to an ice-cold sterile Dounce homogenizer, and lysed with 30 strokes of
a tight fitting pestle. Nuclei were collected by centrifugation at 1600 rpm for 5 min at 4 °C, and the pellet was resuspended in 2 ml of
buffer C (20 mM HEPES (pH 7.9 at 4 °C), 25% (v/v)
glycerol, 420 mM NaCl, 1.5 mM
MgCl2, 0.2 mM EDTA, 0.5 mM
phenylmethanesulfonyl fluoride, 0.5 mM DTT, protease
inhibitors). The nuclear suspension was transferred to a sterile 50-ml
tube, and the proteins were extracted for 1 h at 4 °C with
gentle stirring. The insoluble material was removed by centrifugation
at 14,000 rpm for 10 min at 4 °C, and the extract was dialyzed
against two 100-volume changes of buffer D (20 mM HEPES (pH
7.9 at 4 °C), 20% (v/v) glycerol, 100 mM KCl, 0.2 mM EDTA, 0.5 mM phenylmethanesulfonyl fluoride, 0.5 mM DTT, protease inhibitors). Finally, aliquots of the
nuclear extracts were transferred to silanized tubes and snap frozen in liquid nitrogen. Extracts were stored at 80 °C.
To analyze DNA binding proteins from treated cells, micronuclear
extracts were isolated as described (33). Briefly, subconfluent 35-mm
dishes were starved in 0.5% fetal calf serum/F-12 medium for 12 h, followed by treatment with various agents for 12 h (see the
legend for Fig. 5). Cells were washed twice with ice-cold 1× PBS and
collected by scraping into 1.5 ml of ice-cold 1× PBS. Cells were
collected by centrifugation for 20 s at 14,000 rpm and resuspended
in 100 µl of modified buffer C (17.5 mM HEPES (pH 7.9 at
4 °C), 22% (v/v) glycerol, 368 mM NaCl, 1.3 mM MgCl2, 175 µM EDTA, 0.5%
Triton X-100, 0.5 mM DTT, 0.5 mM
phenylmethanesulfonyl fluoride, 350 µM
Na3VO4, and protease inhibitors) with gentle pipeting. Proteins were eluted for 1 h on ice, and the extract was
cleared by centrifugation at 14,000 rpm for 2 min at 4 °C. Protein
concentrations of the extracts were determined with a modified Bradford
assay (Bio-Rad) and used directly.
Complementary oligonucleotides encompassing the desired binding site
sequences were synthesized locally. All oligonucleotides used were
gel-purified and subsequently annealed in 1× oligo buffer (10 mM Tris·HCl (pH 7.5), 1 mM EDTA, 50 mM KCl) by mixing in a 1:1 molar ratio, heating to 95 °C
for 2 min, 97 °C for 30 s, and cooling to 25 °C over a
period of 2 h. Duplex oligonucleotide probes were then end-labeled
with exo
Klenow (Stratagene, Inc.) and purified over an
S-200 HR size exclusion column (Amersham Pharmacia Biotech). The
specific activity of the probe was estimated and ultimately diluted to
25,000 cpm/0.1 pmol prior to hybridization. Hybridizations were
performed at room temperature for 20 min in a volume of 20 µl, which
consisted of 0.1 pmol of probe (25,000 cpm), 3 µg of bovine serum
albumin (Fisher), 1.25 µg of poly(dI·dC) (Amersham Pharmacia
Biotech), 4 µg nuclear extract, and buffer D. Duplex oligonucleotide
competitors were preincubated with the nuclear extract for 5-10 min at
25 °C. The resulting complexes were resolved on a 4-20% gradient gel (Novex) prerun in 0.375× TBE (33 mM Tris borate (pH
8.3), 75 µM EDTA) at 100 V for 100 min as described (31).
Gels were dried under vacuum and subsequently exposed to Kodak X-Omat film.
The sense strand of each oligonucleotide used for EMSAs are shown in
Table I. To demonstrate the functionality
of CAAT/enhancer-binding protein (C/EBP), thyroid hormone response
element (TRE), and retinoid-like orphan receptor family 1 (ROR1)
competitors, the duplex DNA binding sites were radiolabeled and used to
probe CFK2 nuclear extracts. All competitors were able to form gel
shift complexes (data not shown).
FGFR Expression--
Expression of fgfrs was determined by
reverse transcriptase-polymerase chain reaction as described previously
(34).
| |
RESULTS |
Identification and Localization of Chondrocytic Regulatory
Elements--
FGFR3 is an essential regulator of endochondral
ossification and is expressed in a subset of chondrocytes found within
the developing epiphyseal growth plate (15, 16, 35). The specific expression pattern of fgfr3 suggests that it has
stage-specific effects on the process of chondrocyte maturation. To
examine the regulation of fgfr3 gene expression, two
chondrocytic cell lines, CFK2 and RCJ, were utilized. CFK2 cells and
RCJ cells, both derived from fetal rat calvarial mesenchyme, express
chondrocyte-specific genes and elaborate a cartilage-like matrix (26,
27). CFK2 cells express the fgfr1 (Fig.
1A, lanes
1 and 2), fgfr2 (Fig. 1A,
lane 2), and fgfr3 (Fig.
1A, lane 1) genes, while RCJ cells only express fgfr1 (Fig. 1A, lanes
4 and 5) and fgfr2 (Fig.
1A, lane 5). Neither cell line
expresses fgfr4 (Fig. 1A, lanes
3 and 6). These data demonstrate that expression
of the endogenous fgfr3 gene is regulated in a cell-specific
manner and may reflect differences in the stage of chondrocyte
differentiation.
View larger version (26K):
[in this window]
[in a new window]
|
Fig. 1.
Differential expression of FGF receptors and
the fgfr3 promoter in chondrocytic cell lines.
A, analysis of fgfr expression patterns in
chondrocyte-like cell lines. First strand cDNA was synthesized from
RNA obtained from both the CFK2 (lanes 1-3) and
RCJ (lanes 4-6) cell lines. The kinase domains
of fgfrs 1-4 were amplified simultaneously using a single
oligonucleotide primer pair (34). The resulting mixture of polymerase
chain reaction-amplified products was digested with PstI
(lanes 1 and 4), PvuII
(lanes 2 and 5), and EcoRI
(lanes 3 and 6) to determine the
expression of the fgfr1/fgfr3, fgfr2,
and fgfr4 genes, respectively. Fragments were resolved on
2% agarose gels and visualized by silver staining. Predicted sizes for
PstI digests (lanes 1 and
4) are as follows: fgfr1, 133 and 207 nt;
fgfr3, 176 and 166 nt. Predicted sizes for PvuII
digests (lanes 2 and 5) are as
follows: fgfr1, 195 and 146 nt; fgfr2,
233 and 108 nt. Predicted sizes for EcoRI digests
(lanes 3 and 6) are as follows:
fgfr4, 217 and 124 nt. The arrowhead denotes the
position of PstI-digested fgfr3 cDNA and is
seen in CFK2-derived cDNA (lane 1) but not in
RCJ-derived cDNA (lane 2). The undigested
polymerase chain reaction amplification product in lane 2 also indicates fgfr3 cDNA, which is not
digestible with PvuII; this band is not present in
lane 5, indicating the absence of
fgfr3 expression in RCJ cells. M, 123-base pair molecular
size standards (Life Technologies). B, comparison of
p(2951/27)FR3-luc, p(2311/27)FR3-luc, and p(1537/27)FR3-luc
transcriptional activities in CFK2 (shaded bars,
fgfr3-expressing) and RCJ (open bars,
fgfr3-nonexpressing) chondrocyte-like cell lines. All
bar and column charts show data
representative of at least three independent experiments. Constructs
were transfected in triplicate, and both luciferase and
-galactosidase activities were determined as described under
"Experimental Procedures." Values are plotted as -fold induction
over the empty vector (pGL2-Basic), and -fold induction was calculated
by dividing the mean ± S.D. derived for each construct by the
mean of the pGL2-Basic construct (pGL2-Basic = 1).
|
|
To define the cis-acting sequences that are responsible for
the CFK2-specific expression of the fgfr3 gene, we compared
the activities of various fgfr3 promoter fragments in both
the fgfr3-expressing (CFK2) and nonexpressing (RCJ)
chondrocyte-like cell lines (Fig. 1B). Previous work has
demonstrated that basal promoter elements from the fgfr3
gene function equally well in both cell lines (21). However, by
exploiting cell-specific differences, we demonstrated that the
sequences located between 2311 and 1537 selectively enhanced the
transcription of luciferase reporter constructs in the CFK2 cell line
and failed to alter basal transcriptional activity in the RCJ cell line
(Fig. 1B), thus recapitulating the cell-specific expression
pattern of the endogenous fgfr3 gene in these cell lines.
To verify that the chondrocyte cell-specific region (CSR; nucleotides
2311 to 1537) alone was responsible for the observed CFK2-specific
transcriptional activity, the CSR sequences were multimerized into a
three-copy, head-to-tail array and placed 5' of the Rous sarcoma virus
minimal promoter (pRSV-luc; Ref. 22). The transcriptional activity of
the pCSR-luc reporter construct was examined in both the CFK2 and RCJ
cell lines (Fig. 2). The CSR sequence
elements selectively enhanced the transcriptional activity of the RSV
minimal promoter (230-fold relative to pRSV-luc) in the CFK2 cell line,
while only modestly affecting the transcriptional activity of the RSV
promoter in RCJ cells (25-fold). These data, as well as the data
presented in Fig. 1B, demonstrate that enhancer elements
capable of recapitulating the chondrocytic expression of the
fgfr3 gene in vitro are located between
nucleotides 2311 and 1537. Furthermore, the CSR sequences alone
could confer selective transcriptional activity upon a heterologous,
minimal promoter and do not require additional fgfr3
promoter elements to selectively activate transcription in CFK2
cells.
View larger version (14K):
[in this window]
[in a new window]
|
Fig. 2.
CSR sequences confer selective
transcriptional activity upon the RSV minimal promoter.
Top, schematic representation of heterologous promoter
construct. Bottom, sequences between 2311 and 1537 of
the fgfr3 gene were cloned 5' to the RSV minimal promoter
and were tested for transcription-enhancing activity in CFK2 cells
(shaded bars) and RCJ cells (open bars). Data are
presented as in Fig. 1B; however, -fold induction was
determined relative to the pRSV-luc heterologous promoter construct
(pRSV-luc = 1).
|
|
To identify the chondrocyte-specific regulatory element(s) found within
the 2311 to 1537 region, various pieces of the CSR sequences were
multimerized into three-copy, head-to-tail arrays and placed 5'
relative to the RSV minimal promoter (Fig.
3A). The transcriptional
activity of reporter constructs bearing the 5'-most 50 nucleotides of
the CSR (pCSRa-luc, pCSRc-luc, pCSRe-luc, pCSRf-luc, pCSRg-luc, and
pCSRh-luc) was dramatically enhanced in fgfr3-expressing
CFK2 cells (Fig. 3A), while little or no enhancement of
RSV-mediated transcriptional activity was observed in RCJ cells (data
not shown). Therefore, the position of chondrocytic "enhancer" activity was further refined to nucleotides 2311 and 2263 of the
fgfr3 gene. Analysis of these sequences, using the
MatInspector program (36, 37), identified binding sites for several
known transcription factors (Fig. 3B). Of interest were
putative binding sites for the cAMP response element-binding protein
(CREB), thyroid hormone receptor (T3R), C/EBP, and the ROR1/2, all
components of signaling pathways that are known to affect chondrocyte
development (2, 38-43).
View larger version (25K):
[in this window]
[in a new window]
|
Fig. 3.
Localization of chondrocytic transcriptional
regulatory element(s) in the region 2311 to 1537.
A, the transcriptional activity of deletion constructs
generated from the pCSR-luc reporter were assessed in CFK2 cells. A
schematic diagram of the CSR of the fgfr3 promoter depicts
the following constructs: CSRa, 2311/1947; CSRb, 1968/1534;
CSRc, 2311/2117; CSRd, 2139/1947; CSRe, 2226/2016; CSRf,
2311/2167; CSRg, 2311/2209; and CSRh, 2311/2263. Note that
enhanced transcriptional activity is observed with all constructs
containing the 5'-most 50 nt. Data are presented as in Fig. 2
(pRSV-luc = 1). B, genomic sequence encompassing the
2311 to 2263 chondrocytic region of the FGFR3 gene. Shown both
above and below the sequence are
binding sites for known transcription factors: thyroid hormone response
element (T3R), ROR1/2, CREB, and C/EBP. All sites show a
greater than 90% similarity to known binding sites.
|
|
PTH/PTHrP Receptor Signaling Regulates CSR Activity in
Vitro--
Recent studies have demonstrated that PTH/PTHrP receptor
signaling, like FGFR3 signaling, regulates the process of endochondral bone growth (17-19). fgfr3 is expressed throughout the
proliferative zone, while peak levels of fgfr3 expression
are found in the prehypertrophic region of the epiphyseal growth plate.
PTH/PTHrP receptor is also expressed predominately in prehypertrophic
chondrocytes (19, 44, 45). The overlapping patterns of fgfr3
and PTH/PTHrP receptor expression suggested that PTH/PTHrP receptor
signaling might regulate fgfr3 gene expression in
vivo.
PTH/PTHrP receptor signaling is known to modulate chondrocyte
development, presumably through the activation of cAMP- and/or phospholipase C-associated intracellular signaling cascades (46). Given
the overlap in expression patterns and the presence of a CREB motif in
the CSRh element, it was important to determine whether signals
emanating from the PTH/PTHrP receptor were capable of regulating the
transcriptional activity of the CSRh enhancer element. To mimic
PTH/PTHrP receptor signaling in CFK2 cells, expression vectors encoding
either the wild-type or one of two constitutively active mutant
isoforms of the human PTH/PTHrP receptor (T410P and H223R; Ref. 25),
both associated with Jansen's metaphyseal chondrodysplasia (JMC), were
co-transfected along with pCSRh-luc. Co-expression of the wild-type
PTH/PTHrP receptor, as well as both mutant isoforms, resulted in a
37-40% decrease in CSRh-mediated transcriptional activity (Fig.
4A). Furthermore, the
repression mediated by both the wild-type and T410P mutant PTH/PTHrP
receptors could be enhanced by the addition of 50 nM PTH,
while treatment of the H223R-transfected cells with PTH failed to
augment the observed transcriptional suppression (Fig. 4A,
compare wild type, T410P, and H223R transfections with and without
PTH). Taken together, these data suggest that CFK2 cells express low
levels of a PTHrP-like ligand and that signaling through the PTH/PTHrP
receptor can regulate fgfr3 gene expression through the CSRh
enhancer element.
View larger version (33K):
[in this window]
[in a new window]
|
Fig. 4.
Regulation of CSRh-mediated transcriptional
activity by PTH/PTHrP receptors. A, repression of
pCSRh-luc by co-expression of PTH/PTHrP receptors. pCSRh-luc was
transfected into CFK2 cells along with pcDNA-I, HKrk
(WT), HKrk-T410P (T410P), or HKrk-H223R
(H223R) expression vectors (see "Experimental
Procedures"). Thirty-six hours post-transfection, cells were starved
in Ham's F-12 medium containing 0.5% serum (12 h) prior to
stimulation with or without 50 nM human PTH-(1-34) (12 h).
Lysates were harvested, and data were plotted as percentage of control
(pcDNA-1 vector = 100%). B, regulation of pCRE-luc
transcriptional activity by co-expression of PTH/PTHrP receptors. CFK2
cells were transfected with pCRE-luc, and one of the various expression
vectors as described for A. Data are presented as -fold
induction (vector with or without PTH transfections = 1) and were
calculated by dividing mean ± S.D. derived for each treatment by
the mean of the mock transfection.
|
|
To confirm that PTH/PTHrP receptors were functioning as expected in
CFK2 cells, both wild-type and JMC-associated PTH/PTHrP receptor
expression constructs were co-transfected with a cAMP-responsive (pCRE-luc) reporter gene. Relative to empty vector, co-expression of
the wild-type PTH/PTHrP receptor resulted in a 2.2-fold induction of
CRE-mediated transcriptional activity, while stimulation of wild-type
receptor-expressing cells with 50 nM human PTH-(1-34) resulted in a 10-fold increase in transcriptional activity (Fig. 4B, compare wild-type receptor transfected cells with or
without PTH with vector controls). Unlike the modest stimulation
observed as a result of expression of the wild-type receptor,
co-expression of either the T410P or H223R mutant PTH/PTHrP receptors
resulted in a PTH-independent 5.6- or 6.3-fold increase in CRE-mediated transcriptional activity, respectively (Fig. 4B, compare
T410P and H223R mutant receptor-transfected cells with vector
transfection). Stimulation of the T410P mutant receptor with 50 nM PTH resulted in a significant increase in CRE-mediated
transcriptional activity (Fig. 4B, compare T410P transfected
cells with and without PTH), while treatment of the H223R-transfected
cells with PTH failed to augment CRE-mediated transcriptional activity.
cAMP-mediated Repression of the Chondrocytic Regulatory Element and
Endogenous fgfr3--
To determine whether the PTH/PTHrP
receptor-mediated transcriptional repression of the CSRh enhancer
element depends upon cAMP-mediated signaling pathways,
pCSRh-luc-transfected CFK2 cells were serum-starved for 12 h to
lower background levels of cAMP. Transfected cells were then exposed to
various pharmacological agents known to elevate the intracellular
concentration of cAMP. Activation of adenylate cyclase with 10 µM forskolin (FSK) resulted in a 90% decrease in the
transcriptional activity of pCSRh-luc (Fig.
5A). Moreover, similar
decreases in the CFK2-specific transcriptional activity of pCSRh-luc
were observed when cAMP levels were elevated either directly with
8-bromo-cyclic adenosine 3',5'-monophosphate (8-Br-cAMP; a
cell-permeable cAMP analog) or indirectly through the inhibition
of cAMP phosphodiesterase activity with
3-isobutyl-1-methylxanthine (IBMX) (47) (Fig. 5A, 81 and
95%, respectively).
View larger version (40K):
[in this window]
[in a new window]
|
Fig. 5.
cAMP-mediated repression of FGFR3 gene
expression. A, response of pCSRh-luc to modulation of
cAMP levels. CFK2-transfected cells were starved for 12 h in 0.5%
serum prior to treatment with 0.1% Me2SO
(Mock), 10 µM FSK, 1 mM 8-Br-cAMP,
or 0.5 mM IBMX. Cells were treated for 12 h, and then
both luciferase and -galactosidase activities were determined. Data
are presented as percentage of control as in Fig. 4B.
B, AMP/cAMP dose-response curves. CFK2 cells were
transfected with pCSRh, starved in low serum conditions as described
above, and then treated with varying concentrations of either AMP ( )
or 8-Br-cAMP ( ) for 12 h prior to harvest. C,
cAMP-mediated repression of p(2311/27)FR3-luc.
p(2311/27)FR3-luc-transfected CFK2 (shaded bars) and RCJ (open bars) cells were
starved for 12 h in 0.5% serum prior to treatment with either
0.1% Me2SO (Mock) or 10 µM FSK.
Cells were treated for 12 h prior to the determination of both
luciferase and -galactosidase activities. Data are presented as in
Fig. 4A. D, FSK-induced repression of
CFK2-specific endogenous rat fgfr3 gene expression. Cells
were serum-starved for 12 h in 0.5% serum prior to treatment for
12 h with either 0.1% Me2SO (DMSO) or 10 µM forskolin. RNA was harvested, and the relative levels
of rat FGFR3 mRNA and -actin were determined simultaneously
using RNase protection. The rat FGFR3 probe protects a 291-nt fragment,
and the -actin probe protects a 126-nt fragment.
|
|
To examine the specificity of this transcriptional inhibition, the
regulatory effects of AMP, the noncyclic precursor to cAMP, were
compared with 8 Br-cAMP. The transcriptional activity of pCSRh-luc was
unaffected by as much as 1 mM AMP, while a
dose-dependent repression of CSR-mediated transcriptional
activity was observed with 8-Br-cAMP (Fig. 5B). A 50%
decrease (IC50) in CSRh-mediated transcriptional activity
resulted from treatment with 70 µM 8-Br-cAMP, while
maximal inhibition (approximately 90%) was obtained with 300 µM 8-Br-cAMP. Together, these data demonstrate that the
transcriptional potential of the CSRh enhancer element is regulated by cAMP.
In addition, we examined the transcriptional response of both the
full-length promoter construct (p(2311/27)FR3-luc) and the
endogenous fgfr3 gene to increasing concentrations of cAMP. Treatment of serum-starved CFK2 cells with 10 µM
forskolin specifically repressed CFK2-cell specific transcriptional
activity of p(2311/27)FR3-luc while having little or no effect upon
the basal transcriptional activity of p(2311/27)FR3-luc in RCJ
cells (Fig. 5C). To examine the effects of cAMP on the
expression of the endogenous gene, serum-starved CFK2 cells were
stimulated for 12 h with either 0.1% Me2SO or 10 µM forskolin, and the relative levels of fgfr3 mRNA were determined by RNase protection (Fig. 5D). Band
intensities were quantified, and the decrease in fgfr3
mRNA levels was determined relative to an internal -actin
control. Treatment with Me2SO failed to repress the steady
state levels of fgfr3 mRNA, while treatment with 10 µM forskolin resulted in a 46% decrease in
fgfr3 mRNA levels. A second experiment was performed
following a 24-h induction with forskolin. At this time point, no
change in fgfr3 mRNA levels was observed relative to
-actin. Together, the transcriptional response of the full-length
promoter and of endogenous fgfr3 to forskolin support the
hypothesis that fgfr3 can be regulated by the intracellular
concentration of cAMP.
Protein Kinase A Overexpression Represses CSRh-mediated
Transcription--
cAMP levels can affect gene transcription by
selectively activating PKA (48). Through its activation, PKA has been
shown to augment the transcription of genes that contain a CRE(s) by selectively phosphorylating CREB at Ser-133 (49). Phosphorylated CREB
is then capable of enhancing transcription by interacting with the
CBP/p300 family of transcriptional co-activators (50, 51).
In the inactive form, PKA exists as a tetramer of two catalytic (C)
and two regulatory subunits. Binding of cAMP by the regulatory subunits
results in the dissociation of the tetramer complex and release of the
active C subunit (52). To assess the ability of C overexpression
to mimic the effects of PKA activation, the transcriptional response of
pCRE-luc was examined after co-transfection with the
Zn2+-inducible metallothionine promoter-C (MT-C)
expression vector (23). As expected, co-transfection of MT-C
resulted in a 3-fold increase in the transcriptional activity of
pCRE-luc, while the Zn2+-dependent
overexpression of C led to a 12-fold enhancement in CRE-mediated
transcriptional activity (Fig.
6A, compare vector with and
without Zn2+ with MT-C with and without
Zn2+). Therefore, overexpression of the catalytic domain of
PKA mimics cAMP-mediated transcriptional activation of CRE-containing
genes.
View larger version (13K):
[in this window]
[in a new window]
|
Fig. 6.
Catalytic subunit of protein kinase A
(C-PKA) mimics transcriptional
repression. A, activation of pCRE-luc by overexpression
of C. CFK2 cells were transfected with combinations of pCRE-luc and
either pEV142 (vector) or MT-C expression plasmids. Transfected
cells were starved in Ham's F-12 medium containing 0.5% serum for
12 h prior to stimulation with or without 80 µM
Zn2+ for 16 h. Lysates were harvested, and data were
plotted as -fold induction (untreated vector transfection = 1).
B, repression of pCSRh-luc by co-expression of C. CFK2
cells were transfected with combinations of pCSRh-luc and either pEV142
(vector) or MT-C expression plasmids. Transfected cells
were then treated as described above; however, data are presented as
percentage of control (untreated vector = 100%).
|
|
To determine if PTH/PTHrP receptor-mediated repression of the CSR
sequences also depends on PKA activation, the MT-C expression vector
was co-transfected with the pCSRh-luc reporter construct. Although
treatment of the control vector-transfected cells with Zn2+
resulted in a 27% decrease in transcriptional activity (Fig. 6B, compare vector co-transfections with and without
Zn2+), an additional 50% decrease in CSRh-mediated
transcriptional activity was observed when C activity was induced
with 80 µM Zn2+ (Fig. 6B, compare
MT-C transfections with and without Zn2+ treatment).
These data suggest that the repression of CSRh-mediated transcriptional
activity by cAMP results from the activation of PKA signaling cascades.
Nuclear Factor(s) Interact with 5'-PuGGTCA-3' Core and Regulate the
Transcriptional Potential of the CSRh Sequences--
Recruitment or
stabilization of the transcriptional initiation complex by DNA-binding
proteins and/or associated accessory factors is thought to be the
underlying mechanism that regulates gene expression. To identify the
DNA-protein interaction(s) that direct the observed chondrocytic
transcriptional activity, the CSRh fragment was radiolabeled and used
as a probe in EMSAs. Incubation of radiolabeled CSRh with extracts from
CFK2 cells identified one major complex (Fig.
7A, lane
2); however, a complex with a similar mobility was also
obtained with RCJ nuclear extracts (Fig. 7A, lane
3). To assess the specificity and identity of the
CSRh-binding protein(s) (CSR-BP), nuclear extracts from CFK2 cells were
preincubated with various unlabeled competitors. The CSR-BP·CSRh
complex was completely disrupted by competition with a 10-fold molar
excess of unlabeled probe (Fig. 7B, lanes
7-9), while a C/EBP consensus binding site failed to
compete efficiently for the binding of CSR-BP even at a 100-fold molar
excess (Fig. 7B, lanes 10-12). Significant competition for the binding of CSR-BP was also observed with TRE and ROR1 consensus binding sites (Fig. 7C,
lanes 14-16 and 17-19,
respectively), while, surprisingly, a known CREB binding
site failed to compete effectively even at a 100-fold molar excess
(Fig. 7C, lanes 20-22).
View larger version (22K):
[in this window]
[in a new window]
|
Fig. 7.
CSRh sequences interact with nuclear
proteins. A, DNA/protein interaction with the CSRh
fragment identifies one resolvable complex. EMSAs were performed by
incubating the radiolabeled CSRh fragment with nuclear extracts
isolated from CFK2 (lane 2) and RCJ
(lane 3) cells. The resulting complexes were
resolved on a 4-20% nondenaturing gradient gel. B and
C, analysis of the specificity of the CSR-BP·CSRh complex.
The specificity of this complex was assessed through competition with a
series of defined transcription factor binding sites. Lanes 7-9, "cold" CSRh; lanes 10-12,
C/EBP; lanes 14-16, TRE; lanes 17-19, ROR1; lanes 20-22,
CREB.
|
|
A number of different Zn2+ finger-containing transcription
factors recognize DNA motifs similar to the 5'-PuGGTCA-3' hexamer element found in the CSRh fragment (53). To determine if this motif is
involved in the binding of the CSR-BP complex, nuclear extracts derived
from CFK2 cells were preincubated with several CSRh-like competitors
(Fig. 8A). Significant
competition for the binding of radiolabeled CSRh was observed when CFK2
cell extracts were preincubated with either DM201/2, a duplex DNA
fragment corresponding to the sequences between 2311 and 2276 of
the mouse fgfr3 gene, or DM221/2, a duplex DNA fragment
containing a tetranucleotide substitution outside of the
Zn2+ finger DNA binding motif (Fig. 8B,
lanes 2-4 and 8-10, respectively). However, mutations that disrupt the 5'-PuGGTCA-3' motif (DM223/4) failed to compete for the formation of the CSR-BP·CSRh complex, even
at a 100-fold molar excess (Fig. 8B, lanes
5-7). Identical results were obtained when DM201/2,
DM223/4, and DM221/1 were radiolabeled and used as EMSA probes directly
(data not shown). Incubation of CFK2 nuclear extracts with DM201/2 and
DM221/2 resulted in the formation of a stable DNA-protein complex,
while DM223/4 failed to form a resolvable DNA-protein complex (data not
shown).
View larger version (42K):
[in this window]
[in a new window]
|
Fig. 8.
Mutations in 5'-PuGGTCA-3' core sequence
abolish DNA binding and enhancer activity. A,
sense-strand sequences of duplex DNA fragments used to compete for CSRh
DNA binding activity. S/THR identifies the complement of the hexameric
half-site. DM201/2 corresponds to sequences between 2311 and 2276
of the mouse fgfr3 gene, while mutations in DM223/4
(5'-PuGGTCA-3' core) and DM221/2 (AT-rich sequences) are
underlined. B, competition for binding of
radiolabeled CSRh fragment with DM201/2, DM223/4, and DM221/2 duplex
DNA sequences. Extracts were preincubated with 10-, 50-, or 100-fold
molar excess of unlabeled competitor DNA prior to incubation with
labeled CSRh fragment. The resulting complexes were resolved on a
4-20% nondenaturing gradient gel. C, transcriptional
activity of pmCSRh-luc reporter construct. Mutations corresponding to
those in DM223/4 were introduced in the pCSRh-luc construct to generate
pmCSRh-luc. The transcriptional potential of pmCSRh-luc was then
compared with pCSRh-luc in CFK2 cells. Data are plotted as the
mean ± S.D. of the ratio of luciferase to -galactosidase for
three independent assays. D, CSR-BP·CSRh complex formation
following cAMP induction. CFK2 cells were serum-starved for 12 h
prior to treatment as follows: no treatment, 0.1% Me2SO
(DMSO), 10 µM FSK, or 0.5 mM IBMX.
Modified nuclear extracts (see "Experimental Procedures") were
prepared from the treated cells, and protein concentrations were
quantified. Equal amounts of protein were complexed with the
radiolabeled CSRh fragment and resolved on a 4-20% nondenaturing
gradient gel.
|
|
To determine whether the loss of protein binding activity observed with
the DM223/4 mutations correlated with a loss in CSRh-mediated transcriptional activity, mutations in the Zn2+ finger DNA
binding motif (Fig. 8A, DM223/4) were introduced into pCSRh-luc (pmCSRh-luc), and the resulting transcriptional activity was
examined in CFK2 cells. Disruption of the 5'-PuGGTCA-3' motif resulted
in a 75% decrease in CSRh-mediated transcriptional activity (Fig.
8C). These data suggest that the 5'-PuGGTCA-3' sequence motif regulates the transcriptional activity of the CSRh fragment by
facilitating the formation of a CSR-BP·CSRh transcriptional regulatory complex.
To determine if cAMP levels affect the transcriptional activity of the
CSRh element by regulating the formation of the CSR-BP·CSRh complex,
modified nuclear extracts were prepared from cells treated with 0.1%
Me2SO, 10 µM forskolin, or 0.5 mM
IBMX. Although treatment with either forskolin or IBMX resulted in a
90-95% decrease in transcriptional activity (Fig.
5A), the CSRh binding activity found in CFK2 cells was not
affected by elevations in the concentration of cAMP (Fig.
8D).
The 5'-PuGGTCA-3' Motif Is Not Sufficient for cAMP-mediated
Suppression of the CSRh Element--
To determine whether the
Zn2+ finger binding motif is both necessary and sufficient
for the cAMP-mediated suppression of the CSRh enhancer element, the
transcriptional response to forskolin of the full-length promoter
containing the Zn2+ finger mutation
(pm(2311/27)FR3-luc) was examined. Like p(2311/27)FR3-luc, the transcriptional activity of pm(2311/27)FR3-luc was suppressed by treatment with forskolin, whereas the activity of the promoter lacking the CSR element (p(1537/27)FR3-luc) was largely unaffected (Fig. 9). Despite the marked decrease in
basal activity, pmCSRh-luc also remained responsive to cAMP (Fig. 9).
Together, these results suggest that another CSRh binding activity (or
activities) is required for cAMP-mediated suppression of the CSRh
enhancer element.
View larger version (19K):
[in this window]
[in a new window]
|
Fig. 9.
Binding activity of the CSR-BP is not
necessary for regulation by cAMP. CFK2 cells transfected with the
designated constructs were serum-starved for 12 h in Ham's F-12
medium containing 0.5% serum and subsequently treated with either
0.1% Me2SO (DMSO) or 10 µM FSK
(dissolved in Me2SO). Data are presented as percentage of
control (Me2SO-treated transfections = 100%,
control). p(2311/27)FR3-luc and p(1537/27)FR3-luc are described
in Fig. 1; pCSRh-luc and pmCSRh-luc constructs are described in Fig. 8;
pm(2311/27)FR3-luc contains the 3-base pair substitution
corresponding to the mutation in DM223/4.
|
|
| |
DISCUSSION |
Recently, both gain-of-function and loss-of-function mutations in
fgfr3 have revealed unique roles for this receptor during development (6, 15, 16, 54). Loss-of-function alleles of
fgfr3 lead to an increase in the size of the hypertrophic
zone and the subsequent overgrowth of long bones (15, 16), while gain-of-function mutations in fgfr3 have been genetically
linked to autosomal dominant disorders where both the size and
architecture of the epiphyseal growth plate are altered (8-11).
Together, these observations demonstrate that FGFR3-mediated signaling
is an essential regulator of endochondral ossification.
Previous studies that initially characterized the fgfr3
promoter identified sequences between 220 and +612 of
fgfr3 that were capable of promoting efficient transcription
in vitro, as well as supporting tissue-specific expression
in vivo (21). Nevertheless, these minimal elements failed to
recapitulate the entire tissue-specific expression pattern of
fgfr3 and suggested the existence of other "enhancer"
elements. A chondrocytic "enhancer" capable of recapitulating the
in vitro expression pattern of the endogenous
fgfr3 gene was identified by examining the transcriptional activity of a number of different reporter constructs in
fgfr3-expressing (CFK2) and nonexpressing (RCJ) cell lines.
The sequences found between 2311 and 1537 (CSR) of fgfr3
enhanced the transcriptional activity of the fgfr3 promoter
in CFK2 cells, yet did not affect basal transcriptional activity in RCJ
cells. The inability of the CSR enhancer to stimulate transcriptional
activity in RCJ cells demonstrated that this element is not required
for fgfr3 basal promoter activity and that it functions in a
cell-specific manner. It should also be noted that sequences located 5'
to the CSR suppressed CFK2-specific transcriptional activity; however, the nature of this repression remains to be identified and characterized.
In many cases, transcription factor binding sites are modular and can
confer transcriptional activity upon heterologous promoters. Using the
RSV minimal promoter, sequences between 2311 and 1537 of
fgfr3 were found to selectively enhance the RSV-mediated
transcriptional activity in CFK2 cells, thus demonstrating that these
sequences are both necessary and sufficient for chondrocytic
cell-specific regulation in vitro. Additional deletion
constructs were used to map the chondrocytic "enhancer" activity to
sequences between 2311 and 2263 (referred to as CSRh). Analysis of
this sequence identified overlapping binding sites for two
steroid/thyroid hormone receptor superfamily members and a CREB-like
binding site.
Like FGFR3, both gain-of-function and loss-of-function alleles of the
PTH/PTHrP receptor have been linked to defects in endochondral bone
growth (17, 25, 55). These results demonstrate that PTHrP signaling,
like FGF signaling, regulates endochondral ossification. In
situ hybridization studies have demonstrated that the expression pattern of the PTH/PTHrP receptor and fgfr3 overlap in the
epiphyseal growth plate and suggest that PTHrP signaling may modulate
the chondrocytic expression of fgfr3 in vivo.
Both PTH and PTHrP, through their interaction with a common G
protein-coupled receptor (PTH/PTHrP receptor), activate cAMP and
Ca2+ second messenger signaling pathways by stimulating
adenylate cyclase and/or phospholipase C activity, respectively (46). To determine if PTH/PTHrP receptor signaling could affect the transcriptional activity of the CSRh enhancer element, both wild-type and the constitutively active JMC isoforms of the human PTH/PTHrP receptor were co-transfected with pCSRh-luc. Although
ligand-independent suppression of the CSRh enhancer was observed with
all receptor isoforms, only cells expressing either the wild-type or
the T410P mutant PTH/PTHrP receptors responded to PTH stimulation by
further suppressing the transcriptional activity of the CSR element.
Biochemical characterization of the JMC mutations has demonstrated that
both the T410P and H223R mutations induce the ligand-independent production of cAMP (55); however, PTH/PTHrP receptors harboring the
H223R mutation fail to stimulate inositol phosphate turnover in
response to ligand binding (25). Therefore, these results, as well as
preliminary studies looking at the role of intracellular Ca2+, suggest that maximal repression of the CSRh enhancer
element requires both the cAMP- and
Ca2+-dependent signaling
pathways.2 Such synergistic
"cross-talk" between cAMP and Ca2+ second messenger
signaling cascades may result from a positive feedback loop involving
the activation of Ca2+/calmodulin-stimulated adenylate
cyclases (56). Additional studies will be required to elucidate the
transcriptional role of Ca2+.
Forskolin, a naturally occurring diterpene, elevates intracellular cAMP
levels by directly activating adenylate cyclase. Unlike activation of
the well characterized CREB signaling pathway, forskolin suppressed the
CFK2-specific transcriptional activity of the p(2311/27)FR3-luc and
pCSRh-luc reporter constructs, as well as expression of the endogenous
fgfr3 gene. These observations, as well as the
dose-dependent repression of pCSRh-luc activity by
8-Br-cAMP, demonstrated that the chondrocyte-specific transcriptional
activity of the CSRh enhancer element can be down-regulated by cAMP.
Enhancer elements influence transcription by facilitating and/or
stabilizing the assembly of the transcriptional initiation complex.
Therefore, EMSAs were used to identify potential protein/CSRh DNA
interactions. These experiments identified one DNA-protein complex in
CFK2 nuclear extracts (CSR-BP·CSRh). Proteins in the CSR-BP·CSRh
complex were recognized by steroid/thyroid hormone (S/THR) binding site
(T3R, ROR1, and rOCRE (data not shown)) competitors but did
not interact with known cAMP-responsive consensus binding sites (C/EBP
and CREB), even at a 100-fold molar excess. The interaction of
steroid/thyroid hormone transcription factors with the DNA helix relies
upon the conserved 5'-PuGGTCA-3' hexamer motif (53). Although most of
the S/THRs bind as homo- or heterodimers to variably spaced direct or
indirect repeats of this motif (57), several of the orphan S/THRs have
been shown to bind as monomers to single half-sites (58, 59).
Previously, S/THRs have been implicated in the
cAMP-dependent transcriptional regulation of various
cytochrome P450 (steroid hydroxylase) genes (60). Characterization of
the transcriptional regulation of the P450c17 and P450c21 genes identified cAMP-response elements that differ from the classic CRE-motif (61). These sites resemble the classic 5'-PuGGTCA-3' hexamer
orphan receptor half-site and bind various members of the S/THR
superfamily (SF-1 and COUP-TF (62-64) and NGFI-B (65), respectively).
Together, these results suggest that the factor(s) binding to the CSRh
enhancer element belong to the S/THR superfamily and are not related to
the CREB/CREM/ATF family of basic leucine zipper transcription factors.
Because the CSRh sequence appears only to contain a single half-site,
the effects of elevated cAMP levels and disruption of the 5'-PuGGTCA-3'
sequence motif on CSR-BP·CSRh complex formation were examined. First,
treatment of cells with forskolin does not appear to alter the
formation of the CSR-BP·CSRh complex, suggesting that additional
elements not identified by EMSAs interact with the CSRh sequence.
Second, such factors are required to modulate the cAMP response, and
the action of these elements is independent of CSR-BP·CSRh complex
formation. Third, a mutation that altered three of the six consensus
nucleotides inhibited formation of the CSR-BP·CSRh complex and
significantly reduced the CSRh-mediated transcriptional activation of
pCSRh-luc; however, it does not abolish the cAMP-induced suppression of
the CSRh enhancer element or the full-length promoter. Together, these
results suggest that the orphan receptor half-site is required for
maximal CFK2-specific transcriptional activity, but other undefined
DNA/protein interactions are required for cAMP responsiveness.
Additional experiments will be required to determine which portion of
the CSRh sequence is important for mediating the cAMP response and the
proteins that mediate the response.
The data presented in this study suggest that signaling cascades in the
epiphyseal growth plate capable of modulating the amount of cAMP and
the activity of PKA may affect chondrocyte growth and differentiation
by regulating fgfr3 gene expression in proliferating
chondrocytes. Recently, interactions between the signaling pathways
involving Indian hedgehog and PTHrP have been reported in the growth
plate (17, 45). Coordination of these signals appears to be necessary
to maintain the orderly transit of chondrocytes through the growth
plate. Animals deficient in either PTHrP or the PTHrP receptor have
decreased chondrocyte proliferation (17, 18). Ligand-independent
activating mutations in FGFR3 (G380R, R248C, or K650E) (13, 14) also
decreases chondrocyte proliferation (44,
66).3 Therefore, we
hypothesize that one pathway by which PTHrP can stimulate chondrocyte
proliferation may involve down-regulation of fgfr3
expression. This would link the Indian hedgehog/PTHrP signaling pathway
to the FGFR3 signaling pathway in the epiphyseal growth plate.
Interestingly, recent in vivo studies have demonstrated that
FGFR3 signaling can repress Indian hedgehog activity in the growth
plate (44). This completes a potential feedback loop that may
coordinate endochondral bone growth.
, and
TOP
ABSTRACT
INTRODUCTION
