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Endocrinology Vol. 141, No. 8 2914-2922
Copyright © 2000 by The Endocrine Society

ARTICLES

Nuclear Factor-{kappa}B p50 Is Required for Tumor Necrosis Factor-{alpha}-Induced Colony-Stimulating Factor-1 Gene Expression in Osteoblasts1

Gang-Qing Yao, Ben-hua Sun, Karl L. Insogna2 and Eleanor C. Weir2,3

The Section of Comparative Medicine (G.-Q.Y., E.C.W.), and the Department of Internal Medicine (B.-H.S., K.L.I.), Yale University School of Medicine, New Haven, Connecticut 06520

Address all correspondence and requests for reprints to: Gang-Qing Yao, Section of Comparative Medicine, Yale University School of Medicine, P.O. Box 208016, New Haven, Connecticut 06520-8016. E-mail: gang-qing.yao{at}yale.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Colony-stimulating factor (CSF)-1 is a hematopoietic growth factor that is released by osteoblasts and is recognized to play a critical role in bone remodeling in vivo and in vitro. We have reported that osteoblasts express CSF-1 constitutively and that tumor necrosis factor (TNF)-{alpha}, a potent bone-resorbing agent, increases CSF-1 gene expression by a transcriptional mechanism. In the present study, we report that an NF-{kappa}B site in the CSF-1 promoter is required for TNF-{alpha}-induced CSF-1 expression in osteoblasts. As determined by electrophoretic mobility shift assays, antiserum against the NF-{kappa}B-binding protein, p50, retarded the mobility of the inducible complex, whereas antisera against p52, p65, c-Rel, Rel B, I{kappa}B {alpha}, I{kappa}B {gamma}, and Bcl-3 had no effect. To further confirm that p50 is necessary for TNF-{alpha}-induced CSF-1 expression in osteoblasts, CSF-1 messenger RNA expression from untreated and TNF-{alpha}-treated osteoblasts, prepared from wild-type and p50 knock-out mice, was examined by Northern analysis. CSF-1 messenger RNA was increased by TNF treatment in wild-type mice but not in NF-{kappa}B p50 knock-out mice. Our findings support the conclusion that the NF-{kappa}B subunit p50 is critical for TNF-induced CSF-1 expression in osteoblasts.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
COLONY-STIMULATING factor (CSF)-1 plays an important role in osteoclastogenesis and bone resorption by supporting the proliferation and differentiation of cells in the monocyte/macrophage cell lineage (1, 2, 3, 4). The strongest evidence for CSF-1’s role in bone resorption is that the op/op mouse, which lacks functional CSF-1 because of a mutation in the CSF-1 gene, is devoid of osteoclasts and has an osteopetrotic phenotype caused by impaired bone resorption (5, 6, 7). Treatment of these mutant mice with CSF-1 corrects the defect in bone-remodeling (7, 8).

Studies in vitro have demonstrated that CSF-1 is critical for the proliferation and differentiation of osteoclast progenitors (9, 10), that CSF-1 stimulates bone resorption in the fetal mouse metacarpal assay (11), and that CSF-1 receptors are present on osteoclasts (12, 13). Additionally, we have reported that CSF-1 is the principal colony-stimulating activity released from osteoblasts constitutively and in response to PTH and PTH-related protein (14).

Tumor necrosis factor (TNF)-{alpha} is a multifunctional cytokine, which, in addition to its role in inflammation and immune modulation, is a potent bone-resorbing agent (15, 16, 17). Although the precise mechanism by which TNF stimulates bone resorption is unclear, this action is thought to be attributable, at least in part, to the release of osteoblast-derived factors that stimulate osteoclast progenitor proliferation. TNF-induced bone resorption in vitro is critically dependent on the proliferation of osteoclast progenitors; and, because CSF-1 is necessary for osteoclast precursor proliferation, CSF-1 may play a role in mediating TNF-induced bone resorption (18, 19). We have observed that TNF induces expression of CSF-1 messenger RNA (mRNA) and protein in osteoblasts (14, 20). Though the induction is through a transcriptional mechanism that is not dependent on new protein synthesis, the precise mechanism of transcriptional activation is unknown (20). Yamada et al. (21) studied regulation of the human CSF-1 gene in HL-60 cells and reported that an NF-kB-like protein is involved in TNF-induced CSF-1 gene expression (21). However, which NF-kB family members are involved and whether the same promoter elements and transcription factors are involved in TNF-induced CSF-1 gene expression in osteoblasts are unknown.

NF-kB is a key mediator of TNF-induced regulation of many genes (22, 23, 24, 25). The NF-kB proteins p50 and p52 have recently been shown to be required for osteoblast-mediated osteoclast formation (26, 27). Because osteoblast expression of CSF-1 is required for osteoblasts to support osteoclast formation, we sought to define the cis-elements involved in TNF-induced CSF-1 gene expression in these cells and to determine whether p50 and p52 were involved in mediating this effect.

In the present study, we show that TNF-induced CSF-1 gene expression in primary human osteoblasts and osteoblast-like cells is mediated through activation of NF-{kappa}B and that the NF-{kappa}B subunit, p50, is required for this response.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
TNF, antibodies, and NF-kB oligomers
Human recombinant TNF-{alpha} was purchased from Sigma (St. Louis, MO). Antibodies against NF-{kappa}B p50, p52, p65, c-Rel, and Rel B, as well as antibodies against I{kappa}B family members Bcl-3, I{kappa}B {alpha}, and I{kappa}B ß, were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). A second set of antibodies directed against the NF-{kappa}B p50, p52, and p65 were purchased from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). 22-merNF-{kappa}B consensus (5'-AGT TGA GGG GGC TTT CCC AGG C-3') and mutant oligonucleotides (5'-AGT TGA GGC GGC TTT CCC AGG C-3') were purchased from Santa Cruz Biotechnology, Inc.. An oligonucleotide encompassing the NF-{kappa}B site of the CSF-1 promoter (5'-CAA GGG ACT TTC CCT CCA-'3) was synthesized by the Yale Oligonucleotide Synthesis Lab.

Animal
Breeding pairs of mice homozygous for a targeted disruption of the gene for the NF-{kappa}B p50 (B6, 129 NF-{kappa}B1) and wild-type mice (B6, 129F2) were purchased from The Jackson Laboratory (Bar Harbor, ME).

Cell culture and TNF treatment
Primary human osteoblasts were obtained by explant outgrowth of bone fragments prepared from surgical specimens as reported previously (28). Dr. Mark Horowitz (Department of Orthopaedics, Yale School of Medicine) kindly provided these cells. Primary murine osteoblasts were prepared from calvariae of NF-{kappa}B p50 knock out and wild-type mice by collagenase-dispase digestion, as described previously (14). Primary osteoblasts prepared in this way, the human osteosarcoma cell-line MG63, and the murine osteoblast cell line MC3T3-E1 were maintained in MEM-{alpha} modification (Sigma) containing 10% FBS (Life Technologies, Gaithersburg, MD), penicillin (50 U/ml), and streptomycin (50 µg/ml). Osteoblast cell lines were passaged with trypsin every week and studied at least 3 days post confluence unless otherwise indicated. Primary osteoblasts were passaged with trypsin when confluent and were studied at confluence after the second passage. The human T-lymphoblast cell line, CEM, and the human cervical carcinoma cell line, Hela, were grown and maintained in RPMI1640 (Sigma) containing 10% FBS (Life Technologies), penicillin (50 U/ml), and streptomycin (50 µg/ml). TNF treatments (final concentration, 20 ng/ml) were for 24 h unless otherwise indicated.

Plasmids
The plasmid p-565CAT, a CSF-1 promoter-CAT fusion gene spanning bp -565 to +15 of the human CSF-1 5' flanking region, and seven 5' deleted promoter fragments linked to the CAT gene were kindly provided by Dr. Donald Kufe (Dana-Farber Cancer Institute, Boston, MA). Fragments, containing bp -1239 to +15, -969 to +15, and -719 to +15 of the human CSF-1 promoter region were amplified from human genomic DNA (Promega Corp., Madison, WI) by PCR. The fragments were subcloned into the sacI and HindIII sites of the p{Delta}-CAT vector (21). Double-stranded sequencing was done to confirm the sequence of the inserts.

Site-directed mutagenesis
Site-directed mutagenesis was performed using QuickChange site-directed mutagenesis kit from Stratagene. Mutant NF-{kappa}B binding sites of the p-406CAT plasmid were synthesized with 3-bp substitutions of the wild-type sequence GGGACTTTCCC to mutant TCCACTTTCCC. Positive clones (P-406 mt) were selected by ampicillin, and the mutation was confirmed by sequence analysis.

CAT assay
Osteoblasts in 6-well plates were transiently transfected at 50–60% confluence with 1 µg plasmid DNA using lipofectAMINE (Life Technologies). At 24 h after transfection, cells were cultured in the presence or absence of TNF. Cells were harvested and lysed in 100 µl lysis buffer (Promega Corp.), followed by one cycle of freeze-thawing. CAT activity was determined in 100 µl-reactions containing 50 µl cell extract, 0.025 mCi [14C] chloramphenicol (New England Nuclear, Boston, MA), and 0.25 mg/ml butyryl CoA, for 1 h at 37 C, using a phase extraction CAT assay (29). To control for transfection efficiency, cells were cotransfected with 1 µg human GH plasmid DNA. GH secreted into the culture media by transfected cells was quantitated using an RIA kit (Nichols Institute Diagnostics, San Juan Cappistrano, CA). CAT activity was normalized for transfection efficiency based on GH levels in each sample (29). All transfections and CAT assays were repeated in at least four independent experiments.

PCR amplification
A 93-bp CSF-1 promoter DNA fragment (positions -420 to -328), containing the putative TNF-response element, was amplified using the GeneAmp PCR Kit from Perkin-Elmer Corp. (Branchburg, NJ), according to the recommendations of the manufacturer. Briefly, PCR was performed at a final concentration of 1x PCR buffer, 3 mM MgCl2, 2.5 U taq DNA polymerase (Perkin-Elmer Corp.), 5 mM deoxynucleotide triphosphate, 100 pmol each of 5' and 3' primers (5'-GGTCCGTTTTCTGCTAAG-3' and 5'-TCCAGGCTGATTCAGTG-3') and 1 ng p-565CAT plasmid DNA as template, in a total vol of 100 µl. The reaction mixtures were heated for 2 min at 94 C and amplified in a DNA Thermal Cycler (Perkin-Elmer Corp.). The amplification profile included denaturation at 94 C for 1 min, primer annealing at 60 C for 1 min, and extension at 72 C for 2 min, for 35 cycles, followed by a final 7 min of extension at 72 C. The PCR products were separated by electrophoresis in a 1.8% agarose gel. The 93-bp amplicons were excised from the gel, purified with a QIAquick gel extraction kit (QIAGEN), and stored in aliquots at -20 C.

Electrophoretic mobility shift and supershift assays
The 93-bp CSF-1 promoter DNA fragment containing the putative TNF-response element, and NF-{kappa}B consensus oligomers were end-labeled with 32P {gamma}-ATP and used as probes. The end-labeled DNA (1 ng) was incubated with 5 µg nuclear proteins prepared from untreated and TNF-treated osteoblasts in gel shift binding buffer (Promega Corp.) for 20 min at room temperature. Competition studies were performed by adding a 100-fold molar excess of unlabeled double-stranded DNA competitors. The DNA-protein complexes were analyzed on 4% nondenaturing polyacrylamide gels.

Antibody-mediated supershift analyses of the DNA-protein complexes were performed by using 5 µg nuclear proteins incubated either with 1 µg antibody (for antibody preincubation) or with probe (for antibody postincubation), for 20 min at room temperature, and then probe or antibody, respectively, was added. The mixture was then incubated for an additional 20 min at room temperature, and the reactions were run on 4% nondenaturing polyacrylamide gels for 10–12 h. There were no significant differences between the DNA complexes observed with the antibody preincubation or postincubation protocol. The DNA probe did not interact directly with the antibody (data not shown).

RNA preparation and Northern blot analysis
Cells were grown in T-25 tissue culture flasks and treated with vehicle or TNF. The cells were washed twice with cold PBS and harvested in lysis buffer containing 50 mM Tris HCl (pH 8.0), 140 mM NaCl, 1.5 mM MgCl2, 0.5% NP-40, 1000 U/ml RNasin, and 1 mM dithiothreitol. Total RNA was isolated using RNeasy Mini Kit (Qiagen, Valencia, CA). Twenty micrograms of total RNA was electrophoresed on an agarose/formaldehyde gel and transferred to a nylon membrane (Hybond N; Amersham Pharmacia Biotech, Arlington Heights, IL). Northern hybridization was performed as we have previously described (20). The murine CSF-1 probe was a 1.6-kb 5' fragment of the murine CSF-1 complementary DNA (30). To control for RNA loading in untreated and treated samples, cyclophilin RNA was quantitated as previously described (20). Autoradiograms were quantified by densitometric scanning (Personal Densitometer, SI; Molecular Dynamics, Inc., Sunnyvale, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CAT activity induced by CSF-1 promoter fragments in untreated and TNF-treated osteoblasts
To locate and characterize the cis-acting elements mediating this response, a CSF-1 promoter-CAT fusion gene spanning bp -565 to +15 of the human CSF-1 5' flanking region, and seven 5' deleted promoter fragments linked to the CAT gene, were transiently transfected into MG63 cells. The cells were cultured for 24 h in the presence and absence of TNF. For each construct, TNF-induced CAT activity was determined (Fig. 1Go). In untreated cells, removal of 5' 222 bp (p-343CAT) resulted in a 4-fold increase in CAT activity, compared with activity with the full-length construct. This activity was sustained through deletion to -98 bp, but subsequent deletion to -80 bp eliminated basal CAT activity (Fig. 1Go). TNF treatment caused a 9-fold increase in CAT activity in cells transfected with the p-406 CAT construct. All further deletions between bp -343 and -9 abolished the TNF-induced response. TNF also failed to induce significant CAT activity when the p-565 or p-490 CAT constructs were used, which indicates a repressor located between -406 to -490 bp. In an attempt to identify 5' regulatory elements for this repressor, we cloned the -1239 to +15, -969 to +15, and -719 to +15 bp regions of the human CSF-1 promoter into the sacI and HindIII sites of the p{Delta}-CAT vector (21). However, no TNF induction was observed in cells transfected with these constructs (data not shown).



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  		Figure 1. TNF induces expression of CSF-1 promoter-CAT constructs in MG63 cells. MG63 cells (3 x 105) were transfected with 1 µg of various 5' deletion constructs of the CSF-1 promoter linked to the CAT gene. The cells were cultured for 24 h in the presence or absence of TNF (20 ng/ml) for 24 h after transfection. CAT activity was determined as described in Materials and Methods. Relative CAT activities are expressed as values compared with that obtained with untreated p-565 CAT transfectants. The results represent the mean ± SD of four separate experiments.

 
Binding of constitutive and TNF-induced nuclear proteins to CSF-1 promoter fragments
Because these findings indicated that a TNF-responsive element is located between positions -406 bp and -343 bp of the CSF-1 promoter, a 93-bp CSF-1 promoter DNA fragment (positions -420 to -328) was used in electrophoretic mobility shift assays (EMSAs) to examine protein-DNA interactions occurring within this portion of the promoter. Incubation of the 32P end-labeled 93-bp DNA fragment with nuclear proteins from untreated and TNF-treated MG63 cells resulted in a major retarded band that was significantly increased in intensity by TNF treatment, indicating that TNF increases binding of nuclear protein(s) to this CSF-1 promoter sequence (Fig. 2Go). Coincubation with the unlabeled 93-bp fragment substantially competed the retarded band, confirming the specificity of binding. Because there is an NF-{kappa}B consensus sequence (5'-GGGACT T TCCC-3') on the noncoding strand of the 93-bp fragment (31), competition assays were performed with consensus oligomers for NF-{kappa}B and other known DNA-binding proteins to further define potential binding sites. Coincubation in the presence of NF-{kappa}B consensus oligomers slightly inhibited constitutive binding of nuclear proteins to target DNA and substantially inhibited TNF-induced binding. By contrast, there was no detectable competition with mutated NF-{kappa}B binding sequences or with the Sp1, AP-1, AP-2 binding sequences (Fig. 2Go).



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  		Figure 2. Electrophoretic mobility shift assays of DNA-binding proteins from TNF-stimulated and unstimulated MG63 cells. The CSF-1 promoter 93-bp fragment containing the putative TNF-response element was end-labeled with 32P and used as a probe. Binding reactions were carried out in the absence (lanes 1, 2) or presence of 100-fold molar excess of unlabeled double-stranded DNA competitors: 93-bp fragment (lanes 3 and 4), NF-{kappa}B (lanes 5 and 6), AP-1 (lanes 7 and 8), AP-2 (lanes 9 and 10), Sp1 (lanes 11 and 12), and mutant NF-{kappa}B (lanes 13 and 14). DNA-protein complexes are indicated.

 
Because these findings suggested that an NF-{kappa}B site is involved in TNF-induced CSF-1 gene expression in osteoblasts, a p-406CAT construct with a mutated NF-{kappa}B binding site was synthesized by site-directed mutagenesis and was transfected into MG63 cells. This mutation resulted in complete loss of TNF-induced CAT activity in MG63 cells (Fig. 1Go).

Subunits of NF-kB involved in TNF-induced CSF-1 expression
To assess constitutive and TNF-induced nuclear protein binding to the NF-{kappa}B consensus sequence, EMSA’s were performed using an end-labeled NF-{kappa}B oligomer as probe. As shown in Fig. 3Go, nuclear extracts from untreated MG63 cells contain NF-{kappa}B binding activity that is substantially increased in response to TNF. Coincubation in the presence of a 100-fold molar excess of cold NF-{kappa}B substantially inhibited binding, whereas coincubation with a 100-fold molar excess of mutant NF-{kappa}B had no effect (Fig. 3Go). To further characterize these DNA-binding protein(s), antisera against NF-{kappa}B subunits were used in EMSAs. Antiserum against the p50 subunit substantially retarded the mobility of the constitutive and TNF-induced complex, whereas antisera against p65, c-Rel, and Rel B had no effect (Fig. 4AGo). Because I{kappa}B proteins have also been reported to enhance DNA binding activity of NF-{kappa}B proteins and to activate transcription in some cell types (32), antisera against I{kappa}B {alpha}, I{kappa}B {gamma}, and Bcl-3 were also examined in mobility supershift assays. These antisera had no effect on the mobility of constitutive or TNF-induced nuclear protein binding (Fig. 4BGo).



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  		Figure 3. Competition analysis of specific DNA-protein complexes probed with a NF-{kappa}B consensus oligonucleotide. The NF-{kappa}B consensus oligomer was end-labeled with 32P and used as a probe. The end-labeled DNA (1 ng) was incubated with 5 µg of nuclear proteins prepared from untreated and TNF-treated (20 ng/ml) MG63 cells. Binding reactions were carried out in the absence (lanes 1 and 2), or presence of a 100-fold molar excess of unlabeled double-stranded DNA competitors: mutant NF-{kappa}B (lanes 3 and 4) and NF-{kappa}B (lanes 5 and 6). The DNA-protein complexes were analyzed on 4% nondenaturing polyacrylamide gels and are indicated.

 


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  		Figure 4. Supershift mobility assay with NF-{kappa}B and I{kappa}B antisera. A, Nuclear protein extracts from MG63 cells treated either with vehicle or TNF were incubated with antisera against NF-{kappa}B subunits (anti-p50, lanes 3 and 4; anti-p65, lanes 5 and 6; anti-c-Rel, lanes 7 and 8; anti-Rel B, lanes 9 and 10), subsequent to binding reactions with 32P-labeled NF-kB probe. AB refers to supershifted complexes. B, Nuclear protein extracts from TNF treated or untreated MG63 cells were incubated with antisera against I{kappa}B subunits (anti-I{kappa}B{alpha}, lanes 5 and 6; anti-I{kappa}B{gamma}, lanes 7 and 8; anti-Bcl-3, lanes 9 and 10) before binding reactions with 32P-labeled NF-{kappa}B probe.

 
NF-{kappa}B p50 is involved in TNF-induced CSF-1 expression in primary human osteoblasts
Because MG63 is a transformed cell line, we next extended these studies to primary cultures of human bone cells. We have shown previously that TNF enhances CSF-1 gene expression in nontransformed primary human osteoblasts (14, 20). We therefore determined whether NF-{kappa}B also plays a role in TNF-induced CSF-1 production in primary human osteoblast cultures and, if so, which specific subunits are involved. Consistent with our findings in MG63 cells, TNF increased NF-{kappa}B binding activity in nuclear extracts from primary osteoblasts, and the DNA-protein complex was supershifted by antisera against p50 but not the other NF-{kappa}B subunits, including p52 (Fig. 5Go).



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  		Figure 5. TNF-induced NF-{kappa}B-binding proteins in human primary osteoblasts. Nuclear protein extracts from TNF-treated or untreated human primary osteoblasts were incubated with antisera to the following NF-{kappa}B subunits: anti-p50, lanes 3 and 4; anti-p52, lanes 5 and 6; anti-p65, lanes 7 and 8; anti-C-Rel, lanes 9 and 10; anti-Rel B, lanes 11 and12 subsequent to binding reactions with 32P-labeled NF-kB probe.

 
Comparison of NF-{kappa}B complex induced by TNF in CEM and MG63 cells and binding to NF-{kappa}B consensus and CSF-1 oligos
Results of supershift assays indicated that, in human osteoblasts, TNF induces DNA binding of the NF-{kappa}B p50 subunit, whereas other known NF-{kappa}B subunits could not be identified in the complex. Because the p50-p65 heterodimer is the most common NF-{kappa}B complex involved in transcriptional activation, the absence of a supershifted complex in the EMSA, when using the p65 antisera, could be attributed to a low-affinity antibody. Therefore, nuclear extracts were prepared from Hela cells, in which TNF is known to induce NF-{kappa}B p50/p65 binding (33). As shown in Fig. 6Go, TNF-inducible binding in Hela cells was retarded by antiserum against p50 and p65. This was also the case in the human T-lymphoblast cell line CEM, where TNF-induced NF-{kappa}B binding activity was supershifted by antisera directed against either p50 or p65 (Fig. 6Go). To examine whether time of treatment affected the nature of the TNF-induced NF-{kappa}B complex in osteoblasts, nuclear extracts from MG63 cells, treated with or without TNF for 90 min, were used for EMSAs. Consistent with the findings at 24 h., TNF-inducible binding, after 90 min of treatment, was retarded by antiserum against p50 but not by antisera against p65 (Fig. 6Go).



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  		Figure 6. Supershift mobility assay of untreated and TNF-treated Hela, CEM, and MG63 cells using NF-{kappa}B probes from consensus and CSF-1 promoter sequences. Nuclear protein extracts from 90-min TNF-treated (lanes 2, 4, and 6) or untreated (lanes 1, 3, and 5) Hela, CEM, and MG63 cells were incubated with antisera against NF-{kappa}B subunits P50 (lanes 3 and 4) and p65 (lanes 5 and 6) subsequent to binding reactions with 32P-labeled NF-{kappa}B consensus (top) and NF-{kappa}B CSF-1 (bottom) probes. Supershifted complexes both of p50 and p65 were observed in Hela and CEM, whereas only antisera to p50 supershifted the DNA-protein complex in MG63. There were no significant differences observed in NF-{kappa}B complex binding induced by TNF using the two probes.

 
To determine whether the nature of the NF-kB DNA sequence influenced the nature of the complexes formed, EMSAs using nuclear extracts from TNF-treated CEM, Hela, and MG63 cells were repeated using the NF-{kappa}B CSF-1 sequence. There were no significant differences in the binding complexes observed when either the NF-{kappa}B consensus sequence or the NF-{kappa}B CSF-1 sequence was used (Fig. 6Go).

TNF fails to induce CSF-1 expression in osteoblasts from p50 knock-out mice
To further examine the role of NF-{kappa}B p50 in TNF-induced CSF-1 expression in osteoblasts, primary osteoblasts were prepared from calvariae of 5-day-old wild-type or p50 knock-out mice. RNA isolated from untreated and TNF-treated osteoblasts was examined by Northern analysis. Though osteoblasts from wild-type and knock-out mice constitutively expressed roughly equivalent amounts of CSF-1 mRNA, and (as expected) TNF induced a 3-fold increase in CSF-1 expression in osteoblasts obtained from wild-type mice, this response was largely eliminated in osteoblasts from p50 knock-out mice (Fig. 7Go).



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  		Figure 7. TNF effects on CSF-1 transcript levels in osteoblasts from wild-type and p50 knock-out mice. Confluent, calvarial osteoblasts were treated with vehicle or TNF (20 ng/ml) for 24 h, RNA was harvested, and Northern analysis was performed. Densitometric analysis of three separate experiments revealed a mean 3.0-fold increase in transcript expression in wild-type cells and a 1.2-fold increase in cells obtained from knock-out mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
TNF promotes bone resorption in vitro and in vivo by activating mature osteoclasts and by stimulating the proliferation and differentiation of osteoclast precursors (19, 34). In vivo inhibition of TNF decreases osteoclast formation and bone resorption in estrogen-deficient mice (35, 36). Although the precise mechanisms by which TNF affects osteoclasts is unclear, TNF is recognized to induce osteoblast expression of proosteoclastogenic cytokines, such as IL-1, granulocyte macrophage CSF, IL-6, and PG E2 in osteoblasts (37, 38, 39). CSF-1 is known to be a critical participant in osteoblast-mediated osteoclastogenesis, and we have reported previously that TNF transcriptionally activates the CSF-1 gene in osteoblasts (14, 20). The studies reported here were designed to locate and characterize the cis-acting elements mediating this response and to examine the transcription factors involved. Our results indicate that the -95 to -80 region of the CSF-1 promoter is required for constitutive expression of CSF-1 in osteoblasts and that a TNF-response element is located between positions -406 and -343 upstream of the transcription start site. Additionally, we show TNF-induced CAT activity in cells transfected with p-406, but not p-490 CAT, suggesting that a repressor element may be situated between positions -490 and -406. No TNF-induction of CAT activity was observed by using p-1239 CAT, which suggests that the regulatory element for this repressor is located beyond -1239 bp or in a 3' exon or intron. Repressor elements have been identified in other genes transcriptionally activated by TNF, such as the human 2(I) collagen gene and the plasminogen-activator inhibitor type-2 gene (40, 41). It will be of interest to examine the regulation of the repressor in future studies.

TNF treatment of MG63 cells increased binding of a nuclear protein complex to the -406 and -43 bp regions of the CSF-1 promoter, which could be competed by NF-{kappa}B oligomers but not by consensus sequences for other binding sites. Further, mutation of the NF-{kappa}B site in the CSF-1 promoter region (-377 to -368) blocks TNF-induced CAT activity. These results indicate that the NF-{kappa}B binding site within the CSF-1 promoter is a necessary cis-acting element for TNF-induced CSF-1 gene expression in osteoblasts.

NF-{kappa}B is the prototype of a family of transcription factors (the rel/NF-{kappa}B family) that is ubiquitously expressed, and it regulates expression of a large number of cellular genes involved in growth, development, inflammation, and immunity (42, 43). The activity of NF-{kappa}B family members is tightly controlled by coupling to cytoplasmic inhibitory I{kappa}B proteins (32). Treatment of cells with various signaling molecules results in degradation of I{kappa}B, releasing the bound NF-{kappa}B proteins, which then translocate to the nucleus and activate transcription of target genes (42). Because NF-{kappa}B-dependent activation does not require new protein synthesis, the findings reported here are consistent with our earlier observation that cycloheximide, which inhibits protein synthesis, did not block TNF-induced CSF-1 expression in osteoblasts (14).

Members of the NF-{kappa}B family are dimers composed of subunits that include p50, p52, p65, RelB, and c-Rel, which can form both homo- and heterodimers (40). The data from the supershift assays indicates that, in MG63 cells and primary human osteoblasts, TNF induces DNA binding of the p50 subunit but not of other known members of the NF-{kappa}B family. To further examine whether NF-{kappa}B p50 is necessary for TNF-induced CSF-1 expression in osteoblasts, we studied the effects of TNF on CSF-1 transcript expression in osteoblasts derived from p50 knock-out mice and their wild-type littermates. Our finding that TNF did not induce CSF-1 gene expression in the absence of p50 suggests that p50, functioning either as a homodimer or as a heterodimer with an as-yet unidentified partner, is necessary for TNF-induced CSF-1 expression in osteoblasts. Given that homodimers of p50 are thought to be transcriptionally inactive, because p50 lacks a transcriptional activation domain (43, 44, 45), the possibility that osteoblasts express an as-yet unidentified NF-{kappa}B family member must be considered. Alternatively, it has recently been reported that Bcl-3 may participate with p50 homodimers to induce gene transcription in response to cytokines (46, 47). It may be that such a heterotrimeric complex, in part, explains our findings. The failure to detect Bcl-3 in supershift experiments, however, argues against this; although recent reports indicate that the amount of Bcl-3 present is low and only detected using immunoprecipitation EMSAs (46). Experiments are currently underway to explore these various possibilities.

Cultured osteoblasts, isolated from both wild-type and p50 -/- mice, exhibit similar, low-level CSF-1 transcript expression. This finding is consistent with our promoter deletion analysis in which the -95-+10 bp promoter construct [which corresponds to the -152 to +183bp region in the murine CSF-1 promoter (48)] is sufficient for constitutive expression. The NF-{kappa}B site is located considerably upstream from this region and thus does not seem to be required for basal CSF-1 transcript expression. It has been reported that osteoclastogenesis is normal in p50 -/- mice and, because CSF-1 is required for normal osteoclast formation, it may be that other cytokines can induce CSF-1 expression in these cells through non-p50-dependent pathways.

We have shown that TNF activates NF-{kappa}B p50 in MG 63 cells and human primary osteoblasts. Our findings are consistent with the conclusion that the NF-{kappa}B site located in the human CSF-1 promoter is the cis-element required for TNF-induced CSF-1 expression. Interestingly, Isaacs et al. (49) have reported, using a murine CSF-1 promoter construct, that TNF induces NF-{kappa}B p50/p65 complex formation but does not stimulate the NF-{kappa}B response element in the murine CSF-1 promoter when transfected into the murine stromal cell line ST2. These apparently discrepant results could be explained by that fact that the sequences of the NF-{kappa}B sites in the human and murine promoters are different or that the experiments were performed in different cellular backgrounds. Experiments directly comparing the response in ST2 cells and osteoblasts using both constructs would be of interest.

The NF-{kappa}B subunit p65 (RelA) plays an important role in cell proliferation and apoptosis (25). The NF-{kappa}B p50/p65 heterodimer is thought to be a key regulator of genes involved in responses to infection, inflammation, and stress (50). TNF has been reported to regulate GM-CSF in MG63 cells and primary human osteoblasts and I{kappa}B, through activation of the NF-{kappa}B p50/p65 complex (23). There is little available information, however, on whether the p50/p65 complex is activated by TNF in osteoblasts. IL-1 has been shown to increase NF-{kappa}B binding activity in murine MC3T3 E-1 cells, and the complex can be supershifted by antisera directed against p50 and p65 (51). In the present study, we found that TNF induced p50, but not p65, in MG63 cells and primary human osteoblasts. Similar results were observed in MC3T3 E-1 cells (data not shown). Li et al. (22, 52) reported that TNF, but not IL-1, down-regulates the osteocalcin gene promoter in rat osteoblasts. By supershift analysis, they demonstrated that TNF induced p50 to bind to this negative regulatory element, but they could not identify other NF-{kappa}B subunits in the complex. They concluded that a p50 homodimer might be responsible for the observed effect (22). These finding and ours indicate that TNF may not activate the NF-{kappa}B p50/p65 complex in osteoblasts, although it does so in other cell types (23). Activation of NF-{kappa}B-binding proteins is complex and involves several levels of regulation, including the IKK complex and I{kappa}B family members (53). Different cytokines may induce binding of different NF-{kappa}B family members by influencing one or several of these regulatory steps. Whether or how differential regulation of these molecules underlies the differences in IL-1 and TNF-induced NF-{kappa}B complexes in osteoblasts remains to be clarified.

In summary, we have identified a NF-{kappa}B binding site as the cis-acting element required for TNF-induced CSF-1 expression in osteoblasts. The NF-{kappa}B subunit, p50, is required for this inductive response. This signaling pathway for TNF-dependent CSF-1 gene activation may be cell type-specific and may be one mechanism for tissue-specific actions of TNF in bone.


   Acknowledgments
 
We thank Mark C. Horowitz for supplying human primary osteoblasts, and Sankar Ghosh for helpful discussions.


   Footnotes
 
1 This work was supported by NIH Grants, including DE-12459 (to K.L.I.) and DK-45228 (to K.L.I.), and, in part, by the Yale Core Center for Musculoskeletal Disorders (P30 AR46032; to G.Q.Y. and K.L.I.). Back

2 These authors contributed equally to this work. Back

3 Present address: Quintiles Scotland Ltd., Research Avenue South, Edinburgh, EH14 4AP, Scotland, United Kingdom. Back

Received November 23, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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