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Department of Research and Medicine, Saint Francis Hospital and Medical Center (S.V., S.R., E.C.), Hartford, Connecticut 06105, and University of Connecticut School of Medicine (S.V., E.C.), Farmington, Connecticut 06030
Address all correspondence and requests for reprints to: Samuel Varghese, Ph.D., Department of Research, Saint Francis Hospital and Medical Center, 114 Woodland Street, Hartford, Connecticut 06105-1299. E-mail: svarghes{at}stfranciscare.org
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Introduction |
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Collagenase-1 [matrix metalloproteinase (MMP-1) or fibroblast collagenase], collagenase-2 (MMP-8 or neutrophil collagenase), and collagenase-3 (MMP-13) constitute a small group of proteases within the MMP family that can cleave fibrillar interstitial collagens at neutral pH (12, 13). Collagenase-3 was identified as a novel collagenase in human breast carcinoma and was found to be expressed in certain normal cells, including rat and human osteoblasts and chondrocytes (14, 15). The known rodent interstitial collagenase, previously thought to be rodent collagenase-1, was found to be highly homologous to human collagenase-3 rather than to collagenase-1 and is now considered to be collagenase-3. The level of collagenase-3 is markedly augmented in rodent osteoblasts in the presence of hormones such as PTH, cytokines such as interleukin-1 and -6, and growth factors that promote bone turnover (16, 17, 18, 19, 20). Thus, collagenase-3 may mediate the degradation of collagenous bone matrix and bone resorption caused by selected regulators of bone cells. The participation of collagenase in bone resorption has been implicated by observations demonstrating that inhibition of collagenase activity decreases osteoclastic bone resorption in response to PTH (21, 22). Therefore, elucidation of the molecular mechanisms of collagenase-3 regulation by PTH and other regulators of bone cell function is critical in understanding the molecular events governing bone remodeling. The genomic DNA for rat collagenase-3 has been cloned and analyzed to locate the DNA elements mediating the PTH effect on the collagenase promoter (23, 24).
In previous studies we observed that bFGF stimulates the synthesis of collagenase-3 transcripts and procollagenase secretion in primary cultures of osteoblast-enriched (Ob) cells isolated from fetal rat calvaria. Although bFGF stimulates collagenase-1 and -3 expression in osteoblasts, the mechanisms determining the induction of collagenase-3 have not been determined (20, 25). bFGF acts on the human collagenase-1 gene through a promoter region consisting of activator protein-1 (AP-1)- and polyomavirus enhancer activator-3 (PEA-3)-binding sites, but the two genes are distinct, and these proteases have distinct distributions and possibly functions (25). In this study we investigated the mechanism of regulation of the collagenase-3 gene by bFGF in rat Ob cells and located the DNA regulatory element mediating the growth factor effect by functional analyses and DNA-protein interactions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Nuclear run-off assay
The nuclear run-off assay was performed as previously described
(27). Briefly, nuclei were isolated from Ob cells by Dounce
homogenization (Kontes Co., Vineland, NJ) in Tris buffer containing
0.5% Nonidet P-40 (Sigma, St. Louis, MO). Nascent
transcripts were radiolabeled by incubation of nuclei at room
temperature for 30 min in a reaction buffer containing 250 µCi (800
Ci/mmol) [
-32P]UTP (DuPont/NEN,
Boston, MA); 500 µM ATP, CTP, and GTP; and 150 U RNasin
(Promega Corp., Madison, WI).
[32P]RNA was isolated by treatment with
deoxyribonuclease I (Life Technologies, Inc.) and
proteinase K (Roche Molecular Biochemicals, Indianapolis,
IN), followed by phenol-chloroform extraction and ethanol precipitation
using ammonium acetate. Linearized plasmid DNA containing 1 µg rat
complementary DNA (cDNA) for collagenase-3 (provided by Dr. Cheryl
Quinn, St. Louis University, St. Louis, MO) or
glyceraldehyde-3-phosphate dehydrogenase (GAPD; provided by Dr. Ray Wu,
Cornell University, Ithaca, NY) or plasmid vector pGL2-Basic
(Promega Corp.) DNA was immobilized onto GeneScreen Plus
(DuPont/NEN) membrane using a slot blot apparatus (28, 29). Membranes with a panel of immobilized DNAs were hybridized with
equal counts per min of [32P]RNA from each
sample at 42 C for 72 h and washed in 0.15 M sodium
chloride, 0.015 M sodium citrate (pH 7), and 0.1% SDS at
62 C. Hybridization of nascent transcripts to different DNAs was
visualized by autoradiography and quantified by densitometry.
Recombinant DNA constructs
A rat collagenase-3 genomic DNA clone was provided by Dr. John
J. Jeffrey, Albany Medical College (Albany, NY) (30). Several
collagenase-3 genomic DNA segments with sequences ranging from -721 to
+28 relative to the transcription start site (24) were generated from
the collagenase genomic DNA clone by restriction endonuclease digestion
or DNA amplification using PCR and recloned into pGL2-Basic as outlined
below. To create -721, -452, and -94 to +28 pCaseLUC constructs, the
genomic DNA clone was digested with ApaI, EcoRI,
and NspI, respectively, blunt ended, then digested with
XhoI and ligated into pGL2-Basic at the
SmaI/XhoI sites. All other constructs were made
by PCR using a 5'-sense primer containing the appropriate collagenase
promoter sequence with a KpnI site introduced at the 5'-end
and a 3'-antisense primer, GL primer-2 (Promega Corp.),
that corresponds to a pGL2-Basic sequence downstream of the multiple
cloning site. The 5'-primers used for -185, -142, -107, and -53
pCaseLUC constructs contained collagenase sequence
(underlined) -185 to -168
(5'-GCGGTACCCACTGAAACTAGAGAT-3), -142 to -128
(5'-CGGGTACCTTCTGCCACAAACCA-3'), -107 to -93
(5'-CGGGTACCCCACGTAAGCATGTT-3'), and -53 to -39
(5'-GCGGTACCAGTGGTGACTCATCA-3'), respectively. PCR products
were agarose gel purified, digested with
KpnI/XhoI, and ligated into pGL2-Basic. The -185
APX and -53 APX pCaseLUC constructs were created with the overlap
extension method by PCR using the -185 and -53 pCaseLUC constructs as
templates and sense or antisense primers containing four base
substitutions to mutate the AP-1 site TGACTCA at -42 to -48 to
TGtgTtg (lowercase bases correspond to base substitutions)
(31). The mutated PCR products generated were purified on an agarose
gel, digested with KpnI/XhoI, and ligated into
pGL2-Basic. A recombinant with a heterologous promoter and three
collagenase AP-1 sites, designated pSV40LUC-3AP, was constructed by
ligating annealed oligonucleotides that correspond to three tandem
repeats of AP-1 consensus sequence (underlined) with an
upstream BamHI recognition site for screening (sense strand
oligonucleotide sequence,
5'-GGATCCTGACTCATGACTCATGACTCAC-3'; antisense strand
oligonucleotide sequence,
5'-TCGAGTGAGTCATGAGTCATGAGTCAGGATCC-3') into the
SmaI/XhoI digested pGL2-Control (Promega Corp.), a luciferase reporter driven by simian virus 40 (SV40)
promoter. The orientation and nucleotide (nt) sequence of the inserts
of the recombinants were confirmed by restriction endonuclease mapping
and DNA sequence analysis. A ß-galactosidase expression construct
using cytomegalovirus immediate early gene promoter (pCMV) was obtained
from CLONTECH Laboratories, Inc. (Palo Alto, CA), and used
for cotransfection to determine transfection efficiency.
Transient transfections and reporter gene assays
The recombinant plasmids used for transfection were purified
either by cesium chloride (Life Technologies, Inc.)
density gradient centrifugation or by a plasmid purification kit (5
Prime
3 Prime, Boulder, CO) (32). Subconfluent Ob cells were
transiently transfected by calcium phosphate-DNA coprecipitation as
previously described (27). Each independent culture was transfected
with 5 µg pCaseLUC and 1 µg pCMV constructs. Cells were grown to
confluence, serum deprived, and exposed to control DMEM or bFGF for
2–16 h. Cells were harvested in 125 µl reporter lysis buffer
(Promega Corp.) according to the manufacturer’s
instructions and assayed for luciferase and ß-galactosidase
activities using an Optocomp II luminometer (MGM Instruments, Hamden,
CT) as previously described (33). To correct for variations in
transfection efficiency, luciferase activity was normalized for changes
in ß-galactosidase activity. In each transfection experiment with
pCaseLUC construct(s), a group of cultures was transfected with
pGL2-Basic (promoterless construct) as a control for background
activity, and the effect of bFGF on pCaseLUC constructs was corrected
for the effect on pGL2-Basic. In transfection experiments with
pSV40LUC-3AP, a group of cultures was transfected with pGL2-Control
(parental heterologous promoter construct), and the effect of bFGF on
pSV40LUC-3AP was corrected for the effect on pGL2-Control.
Electrophoretic mobility shift assay (EMSA)
Preparation of the nuclear extract and EMSA were performed as
previously described (34). Briefly, nuclei were isolated from control
or bFGF-treated Ob cells by lysis using 0.5% Nonidet P-40 followed by
centrifugation. To prepare the nuclear extract, nuclei were incubated
for 1 h at 4 C in a buffer consisting of 20 mM HEPES,
0.2 mM EDTA, 0.4 M sodium chloride, 0.5
mM dithiothreitol, 1 mM
phenylmethylsulfonylfluoride, 10% glycerol (all from
Sigma), 0.1 mM antipain, 2.5 µg/µl
pepstatin, 2.5 µg/µl aprotinin, and 0.1 mM leupeptin
(all peptide protease inhibitors from Calbiochem, San
Diego, CA). After incubation, the supernatant was collected by
centrifugation and stored at -70 C until use. The protein
concentration of the nuclear extract was measured using the DC protein
assay kit (Bio-Rad Laboratories, Inc., Hercules, CA)
(35).
Sense and antisense oligonucleotides ranging in size from 20–30 nt,
corresponding to various regions of the collagenase promoter, were
custom synthesized by Life Technologies, Inc., and
annealed in the presence of 50 mM sodium chloride. The
annealed DNA was used either as probe after radiolabeling or as cold
competitor DNA. To generate the radiolabeled probe, DNA was end labeled
using [
-32P]ATP (DuPont/NEN)
and T4 polynucleotide kinase (Life Technologies, Inc.) and
was purified through a Sephadex G-50 (Pharmacia Biotech,
Piscataway, NJ) gel filtration column. The nuclear extract (8–12 µg)
was sequentially incubated at room temperature with 1 µg double
stranded poly(dI-dC) (Pharmacia Biotech) for 15 min, with
or without a 100-fold excess of cold competitor DNA for 15 min and 100
fmol radiolabeled double stranded DNA probe (
100,000 cpm) for 15
min. For antibody supershift assays, the second incubation was carried
out with 2–4 µl specific antibodies raised against different
transcription factors (Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA) for 30 min to 1 h. The salt concentration in the
final incubation step was 90–140 mM. Glycerol was added to
a final concentration of 10% after the incubation, and the protein-DNA
complexes were resolved at 4 C in a 13-cm long 4% native
polyacrylamide gel. In experiments to resolve the nonsupershifted
DNA-protein complexes, the electrophoresis was performed at 120 V
(constant voltage) for approximately 3 h (until the bromophenol
dye migrated to 70% of the length of the gel). In experiments to
resolve the supershifts with antibodies, the electrophoresis was
performed at 120 V for more than 4 h (until the xylene cyanol dye
migrated to >70% of the length of the gel). The gel was dried at 80 C
under vacuum and autoradiographed to visualize the protein-DNA
complexes.
Statistical methods
Data for collagenase promoter activity are presented as the
mean ± SEM. Statistical differences were determined
by one-way ANOVA (36).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). bFGF at 0.6
nM for 4 h stimulated the rate of transcription by
3-fold (data not shown).
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). To delineate the DNA element(s)
mediating the response to bFGF, additional deletion constructs,
pCaseLUC -452, -185, 142, -107, -94, and -53, were tested, and
their activities after treatment with bFGF at 0.6 nM for
4 h were determined (Fig. 3). The
stimulatory effect of bFGF on the collagenase promoter was retained by
all constructs with a promoter sequence up to -53 nt, indicating that
putative regulatory elements located downstream of -53 nt in the
collagenase-3 promoter mediate the effect of bFGF. As the pCaseLUC -53
construct contains an AP-1 consensus site at -42 to -48 nt, AP-1
mutant constructs pCaseLUC -185 APX and -53 APX, were tested (Fig. 4). bFGF at 0.6 nM for 4
h stimulated the promoter activity of wild-type constructs by 2- to
4-fold, but not that of pCaseLUC -185 APX and -53 APX, indicating
that the AP-1 site at -42 to -48 mediates the response of
collagenase-3 promoter to bFGF. To study the effect of bFGF on the AP-1
site in the context of a heterologous promoter, we tested the promoter
activity of pSV40LUC-3AP, a reporter construct with the tandem repeats
of three AP-1 consensus sequences. Treatment of Ob cells, transiently
transfected with pSV40LUC-3AP, with bFGF at 0.6 nM for
4 h stimulated the promoter activity by 1.73 ± 0.13-fold
(mean ± SEM; n = 6).
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Exposure of Ob cells to bFGF at 0.6 nM for 15 min to 4
h caused the formation of a specific DNA-protein complex (Fig. 5). The specific DNA-protein complex was
distinguished from nonspecific complexes by its disappearance in EMSA
performed in the presence of excess unlabeled AP-1 oligonucleotide as a
competitor. Treatment of Ob cells with bFGF at 0.06–6.6 nM
for 2 h increased the intensity of the DNA-protein complex by 3-
to 4-fold (data not shown). To determine whether the DNA-protein
complex is an AP-1 complex, EMSA was performed using nuclear extracts
from cultures with and without exposure to bFGF for 2 h in the
presence of excess unlabeled APX, PEA-3, and core binding factor-1
(CBFA-1) oligonucleotides or of antibodies against the Jun and Fos
families of transcription factors (Table 1 and Fig. 6). The DNA-protein complex was blocked
by excess AP-1, but not by excess APX oligonucleotide, indicating that
its formation requires an intact AP-1 site. The presence of excess
PEA-3 and CBFA-1 oligonucleotides did not modify the DNA-protein
complex, suggesting that the nuclear factors interacting with these
regulatory elements do not interfere with complex formation. The
DNA-protein complexes were supershifted by antibodies against Jun and
Fos families, confirming the identity of this complex as an AP-1
complex consisting of transcription factors of Jun and Fos families.
There was no residual binding of nuclear factor(s) to the AP-1
sequence, when the nuclear extract was incubated with pooled antibodies
against Jun and Fos families, suggesting that the DNA-protein complex
formed by the collagenase AP-1 site did not include additional related
factor(s) that can directly bind the AP-1 sequence, as shown in the
case of collagenase-3 regulation by PTH (23). The Jun family consists
of c-Jun, JunB, and JunD, and the Fos family consists of c-Fos, FosB,
Fra1, and Fra2 proteins (37). To establish the identity of the members
of the Jun and Fos families that constitute the AP-1 complex in control
and bFGF-treated Ob cells, supershift EMSA was performed using nuclear
extracts from cultures exposed to bFGF for 2 h and antibodies
raised against each member of the Jun and Fos families (Fig. 7). The AP-1 complex from control
cultures was supershifted primarily by a JunD antibody, whereas the
complex from cultures exposed to bFGF was supershifted by antibodies
against c-Jun, JunB, JunD, c-Fos, FosB, and Fra2, suggesting that bFGF
stimulates the formation of a Jun-Fos heterodimer in the AP-1 site of
the collagenase-3 promoter.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Hormones and growth factors are known to regulate gene expression through AP-1 sites (37). The AP-1 sequence binds to members of the Jun and Fos families of transcription factors. These nuclear proteins interact with the AP-1 site as Jun homodimers or Jun-Fos heterodimers formed by protein dimerization through their leucine zipper motifs. These protein dimers can recruit other nuclear proteins, such as glucocorticoid receptors, to AP-1 sites through protein-protein interactions (39, 40, 41). Therefore, a variety of extracellular stimuli can mediate their cellular effects through the AP-1 sequence, making it one of the versatile DNA elements mediating gene transcription. bFGF receptors, upon activation by ligand binding, elicit multiple signal transduction pathways, leading to regulation of different target genes (42). In our study the DNA-protein interactions between the collagenase-3 AP-1 site and the Jun and Fos family members are altered by bFGF. In control cultures, the collagenase AP-1 site is bound primarily to JunD, suggesting that the AP-1 complex in unstimulated cells contains Jun homodimers. Exposure to bFGF causes a marked increase in the binding of several members of the Jun and Fos families to the collagenase AP-1 site, suggesting that the AP-1 complex in the treated cultures may consist of Jun-Fos heterodimers. Wang et al. and Gack et al. observed that collagenase synthesis is induced in various tissues of transgenic animals overexpressing c-Fos, or c-Jun and c-Fos, suggesting that an increase in c-Fos level can stimulate collagenase expression (43, 44). The specific roles of different Jun and Fos proteins in modulating the effects of bFGF on the regulation of collagenase-3 and other bFGF-responsive genes are not known. Depending on its composition, the AP-1 complex can differentially interact with other nuclear factors, such as steroid hormone receptors. Therefore, we speculate that an increase in multiple Jun and Fos proteins by bFGF will provide versatility to the AP-1 complex so that it can integrate signals from other systemic and local agents through diverse protein-protein interactions. Further studies in which specific Jun and Fos proteins are overexpressed or disrupted in an osteoblast-specific manner will be necessary to define the specific roles of these proteins in mediating the effects of bFGF and other regulators of bone remodeling in the skeletal tissue.
Collagenase-3 is constitutively synthesized by osteoblasts, and its synthesis is augmented by a number of regulators of bone resorption, such as PTH and interleukin-1 (16, 17, 18, 19). Unlike osteoblasts, osteoclasts do not express detectable levels of collagenase-3 (45, 46). It is apparent that the primary role of collagenase-3 in bone is to cleave intact collagen fibrils, generating collagen fragments that are susceptible to further digestion by other proteases, such as gelatinases (47). A number of independent studies suggest that the activation of collagenase in bone cells is associated with increased bone resorption (21, 22, 45, 46). Collagenase-3 may also regulate aspects of bone formation, as implicated by the observation that protease can cleave insulin-like growth factor-binding protein-5, generating peptide fragments that can enhance the effects of insulin-like growth factor on bone formation (48).
In conclusion, bFGF stimulates transcription of the collagenase-3 gene. The transcriptional effect of bFGF is mediated through an AP-1 site in the collagenase-3 promoter by the induction or activation of the Jun and Fos families of transcription factors. The stimulation of collagenase-3 synthesis by bFGF may be critical in mediating the actions of this growth factor in bone remodeling.
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Acknowledgments |
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Footnotes |
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Received September 9, 1999.
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