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RESEARCH REPORT |
-induced IL-6 Production in MG-63 Human Osteoblast-like Cells
1 Formerly Dept. of Oral Biology, UNMC, and presently Dept. of Orthodontics, The Ohio State University, Columbus, OH;
2 formerly Dept. of Oral Biology, UNMC, and presently in private practice, Roseburg, OR;
3 Dept. of Oral Biology, UNMC, 40th & Holdrege Sts., Lincoln, NE 68583;
*corresponding author, skoka{at}unmc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF-) stimulates osteoblast production of interleukin-6 (IL-6), an inflammatory cytokine implicated in osteoclastic bone resorption. Therefore, we tested the hypothesis that TNF--induced IL-6 production in MG-63 osteosarcoma cells occurs via the p38 mitogen-activated protein kinase (MAPK) pathway. TNF- activated p38 MAPK and stimulated IL-6 secretion by MG-63 cells, and pre-incubation of cells with the p38 MAPK inhibitor abrogated TNF--dependent IL-6 secretion. Transfection of IL-6 full-length and 5'-deletion gene promoter reporter constructs indicated that p38 MAPK activation by TNF- enhanced IL-6 gene expression, and that the p38 MAPK-responsive region resided in the proximal 260-bp segment. Transfection of NF
B and C/EBPß-sensitive reporter promoter constructs demonstrated that NFB activity was enhanced and that constitutive C/EBPß was inhibited by TNF-, with both effects being p38 MAPK-dependent. In conclusion, although p38 MAPK activation by TNF- stimulates IL-6 secretion by MG-63 cells, it has opposing effects on c/EBPß and NFB activity.
KEY WORDS: map kinase • interleukin-6 • osteoblast • inflammation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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) is a pro-inflammatory cytokine found in increased concentrations in inflamed tissues, such as those encountered in arthritis and periodontal disease, and has been found to stimulate bone resorption (Thomson et al., 1987; Ishimi et al., 1990). At sites of inflammation, osteoblasts of the periosteum react to these inflammatory mediators and consequently produce the cytokine interleukin-6 (IL-6) (Manolagas, 1995). Interleukin-6 is a multifunctional cytokine involved in osteoclast recruitment and differentiation into mature osteoclasts. Osteoblast-derived IL-6, in particular, is crucial to bone remodeling (Chambers and Fuller, 1985; Fuller et al., 1991), since excess IL-6 production predisposes to increased osteoclast number (Ishimi et al., 1990; Rozen et al., 2000).
The mitogen-activated protein kinase (MAPK) family of proteins is involved in intracellular signaling pathways, and three major families of MAPKs have been described in higher eukaryotes (Latchman, 1998). The effects of MAPK activation are varied and include cell proliferation, differentiation, and secretion of cytokines and growth factors (Thomson et al., 1999). The three MAPK families are: (1) the ERK (extracellular signal-related kinase) pathway; (2) the JNK/SAPK (c-Jun NH2-terminal kinase/stress-activated protein kinase) pathway; and (3) the p38 MAPK pathway. To be activated, ERK, JNK/SAPK, and p38 MAPK must be phosphorylated by dual-specificity kinases (MAP Kinase Kinases) on threonine and tyrosine residues within a highly conserved phosphorylation motif T-X-Y, where X is E for ERK, P for JNK/SAPK, and G for p38 MAPK (Latchman, 1998). In response to various stimuli, osteoblasts utilize MAPK-dependent pathways to modulate immediate early-gene induction (Siddhanti et al., 1995; Verheijen and Defize, 1995; Chaudhary and Avioli, 1997,1998;Miyazawa et al., 1998; Miwa et al., 1999). Previous reports suggest that TNF- stimulates ERK, JNK, and/or p38 MAPK to differing degrees, depending upon the cell type investigated and the nature of the stimulation (Vanden Berghe et al., 2000.). In general, the ERK cascade preferentially regulates cell growth and differentiation, while the JNK and p38 MAPK cascades are associated more often with stress responses such as inflammation. However, the role of TNF--activated p38 MAPK in IL-6 production by osteoblasts of human origin is poorly understood.
The majority of evidence regarding IL-6 gene expression indicates that the transcription factor NFB is the principal regulator of transcription (Vanden Berghe et al., 2000). However, in certain cell types, the activity of the transcription factors C/EBPß and activator protein-1 (AP-1) also influences IL-6 gene expression (Akira and Kishimoto, 1992) (Fig. 1
). A variety of transcription factors is initially activated as a result of MAPK pathways, and TNF-, again depending upon cell type, can activate one or more MAPK pathways. Therefore, the p38 MAPK pathway, its activation by TNF- in human osteoblast-like cells, and its effect on IL-6 production and on transcription factors that modulate IL-6 gene expression form the focus of this study.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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was purchased from Pharmingen, Inc. (San Diego, CA, USA). All hIL-6 enzyme-linked immunosorbent assay (ELISA) reagents were purchased from Pharmingen (San Diego, CA, USA), except for 2,2'azino-bis-(3-ethylbenzthiazoline-sulfonic acid) (ABTS) and hydrogen peroxide (Sigma).
IL-6 Secretion
MG-63 cells were plated in 16-mm plastic wells (24-well plates) at a density of 5 x 104 cells/mL per well and incubated overnight. Media were removed and replaced with 1 mL of RPMI 1640 with 0.1% FBS and with various concentrations of TNF- and incubated for 24 or 48 hrs. For experiments involving inhibitor, cells were plated and incubated overnight (same as above) prior to the replacement of media with 1 mL of RPMI 1640 with 0.1% FBS and inhibitors. Cells were incubated for 60 min with various concentrations of SB203580 HCl (10, 5, and 1 µM) prior to stimulation with TNF- (10 ng/mL) for 24 hrs. Supernatants were collected and stored at -70°C until IL-6 enzyme-linked immunosorbent assay.
ELISA for Human IL-6
Briefly, 96-well plates were coated with 50 µL of anti-cytokine capture antibody (2 µg/mL) and incubated overnight at 4°C. After being washed with PBS/Tween and blocked with PBS/10% FBS, 100 µL of experimental supernatants or standards were plated and incubated overnight at 4°C. After being washed, 100 µL of biotinylated anti-cytokine detecting antibody (1 µg/mL) were added and allowed to incubate for 1 hr at room temperature. Plates were washed prior to the addition of 100 µL of streptavidin-horseradish peroxidase enzyme and incubation at room temperature for 30 min. After being washed, 100 µL of ABTS substrate solution supplemented with hydrogen peroxide were added and incubated at room temperature for 30-45 min prior to optical density readings at 405 nm referenced to 490 nm. For each experimental culture well, duplicate ELISA readings were obtained. Sample IL-6 concentrations were interpolated from a linear fit standard curve calculated from known values of IL-6 ranging from 31 pg/mL to 2000 pg/mL.
Activation of p38 MAPK by TNF-
MG-63 cells were plated in 35-mm plastic wells at a density of 3 x 105 cells/mL per well and allowed to attach overnight prior to a 24-hour serum starvation. Media were removed and replaced with 1 mL of RPMI 1640 with 0.1% FBS and with TNF- (20 ng/mL) and incubated for 0, 5, 10, 15, 30, and 45 min. To confirm inhibition of p38 MAPK phosphorylation by SB203580 HCl, we plated MG-63 cells and incubated them overnight (same as above) prior to replacing media with 1 mL of RPMI 1640 with 0.1% FBS and inhibitor. Cells were incubated for 60 min with SB203580 HCl (10 µM) prior to stimulation with TNF- (20 ng/mL) for 5 min.
Western Blotting
We placed plates on liquid nitrogen for 1 min to snap-freeze cells, prior to adding 400 µL of lysis buffer (50 mM HEPES, pH 7.8, 1% Triton, 1 mM EDTA, 30 mM sodium pyrophosphate, 1 mM sodium vanadate, 10 mM sodium fluoride, 1 µM phenylmethylsulfonyl fluoride, 20 ng/mL aprotinin). Lysates were vortexed and centrifuged to "pellet" detergent-insoluble cell debris. Protein content of lysates was determined by means of the BCA Protein Assay (Pierce, Rockford, IL, USA), and 4x SDS sample buffer was added to a volume containing approximately 200 µg of protein before samples were boiled at 100°C for 5 min. Electrophoresis was performed in 10% SDS-PAGE gels prior to transfer to polyvinylidene difluoride membranes for 1 hr at 40 V and 200 mA (transfer buffer: 15 mM Tris, 120 mM glycine, 20% methanol). Membranes were blocked for 1 hr in 3% bovine serum albumin (BSA) in Tris-Buffered Saline/Tween (TBST) (10 mM Tris, pH 8.0, 150 mM NaCl, 0.05% Tween). Membranes were immunoblotted overnight (1:800 in 5% BSA in TBST) with phospho-specific mouse antibody for p38 MAPK (p-p38) (New England BioLabs, Beverly, MA, USA). After being thoroughly washed in TBST, membranes were blotted with alkaline phosphatase-linked secondary goat anti-mouse antibody (New England BioLabs) (1:1000 in TBST) for 1 hr prior to color development with appropriate substrate (nitroblue tetrazolium [0.165 mg/mL] and 5'bromo-4'chloro-3'indolyl phosphate [0.165 mg/mL]) in buffer (100 mM Tris, pH 9.5, 100 mM NaCl, 5 mM MgCl2). Lysates were also blotted for total p38 MAPK (New England Biolabs) content for verification of equal amounts of p38 MAPK protein levels in each sample (data not shown). Lysates from serum-starved cultures constituted negative controls.
Analysis of IL-6 Gene Promoter and NFB and C/EBPß Activity
The full-length human IL-6 gene promoter (phIL-6) attached to the chloramphenicol transferase reporter gene was a generous gift of Dr. Stavros Manolagas (University of Arkansas). A 1.17-Kb fragment of phIL-6 spanning -1160 to +10 (relative to the transcription start site) was amplified by polymerase chain-reaction and subcloned into the firefly luciferase reporter gene-containing pGL-3 Basic vector (Promega, Madison, WI, USA). The resulting construct was termed phIL-6WT. Included in the anti-sense primer (5'-ATCTCGAGGGC AGAATGAGCCTCAGAC-3') sequence was a XhoI restriction site, and a SacI restriction site was included in the sense primer (5'-ATGAGCTCGGATCCTCCTGCAAGAGACA-3'). Two 5'-deletion constructs were generated to create –490 to +10 (hereafter phIL-6A) and a –260 to +10 (hereafter phIL-6B) promoter-reporter fragments. The same anti-sense primer was used to fabricate these 5'-deletion constructs. The sense primer used to generate the phIL-6A fragment was 5'-AGAGCTCGATGGAGTCAGAGGAAACTCAG-3'. For fabrication of phIL-6B, the sense primer was 5'-GCGAGCTCAGAAAGTAAAGGAAGAGTGGTT-3'. Both of these primers also contained a SacI restriction site. The C/EBPß-sensitive promoter was generated as follows to include 4 C/EBPß binding sites. A single primer set was generated with the following sense sequence: 5'ATAGAGCTCATACGTCACATTGCACAATCATAGAGCTCATTACGTCACATTGCACAATCATACAATACGTCACATTGCACAATCATTCATAACGTCACATTGCACAATCTAATCTCGAGTAA-3'. The anti-sense primer was as follows: 5'ATAC TCGAGGATTGTGCAATGTGACGTTTACTCGAGATTAGATTGTGCAATGTGACGTTATGAATGATTGTGCAATGTGACGTATTGTATGATTGTGCAATGTGACGTAATGAGCTCTAT-3'. The underlined areas correspond to the C/EBPß binding site of the human IL-6 gene promoter. At the distal ends of the primers, a SacI restriction site was included, and a XhoI site was included at the proximal ends of the primers. After annealing and SacI/XhoI digestion, the fragment was ligated into pGL-3 per manufacturer's recommendations. Sequences for the phIL-6 constructs and the C/EBPß construct were verified. The NFB-luciferase construct was utilized as previously described (Pahan et al., 2000).
Transfection
MG-63 cells were plated in 35-mm plastic wells at a density of 4 x 105 cells/mL per well and incubated overnight in RPMI 1640 plus 10% FBS and antibiotics. Immediately prior to transfection, cells were washed 2 times in PBS. Cells in each well were transfected by means of 1-2 µg of plasmid DNA, 30 ng of control vector pRL-sv40 (Renilla luciferase), and 16 µg (8 µL) of LipofectAMINE reagent (Life Technologies, Inc., Gaithersburg, MD, USA) per manufacturer's recommendations. After 4-5 hrs, an equal volume of 20% FBS medium was added for an additional 16 hrs. Media were removed, and cells were incubated in serum-free media for 8 hrs, prior to replacement of media with 2 mL of media representative of experimental conditions (TNF with/without pre-incubation with SB203580 HCl for 60 min). After 24 hrs of TNF- exposure, cells were washed in pre-cooled PBS and subjected to lysis in 400 µL of passive lysis buffer (Promega). Lysates were frozen, thawed, vortexed for 15 sec, and centrifuged for 20 sec at 10,000 x g. A 20-µL quantity of supernatant was mixed with 100 µL of luciferase assay buffer, and luciferase activity was measured as light output (10 sec) by a Turner Designs Luminometer Model TD 20/20 (Turner Designs, Sunnyvale, CA, USA). Subsequently, the Renilla luciferase activity was estimated after the addition of 100 µL of Stop and Glo reagent (Promega), and light output (10 sec) was measured separately. We used the Renilla activity to standardize the results for transfection efficiency. Each condition was run in triplicate, and the experiments were repeated at least two more times.
Statistics
We used analysis of variance to test whether TNF concentration and/or pre-incubation with inhibitor affected IL-6 secretion. For statistical analysis of luciferase activity, we used the Kruskal-Wallis test (for non-parametric data) to determine the effect of treatment condition (TNF- concentration/inhibitor) on the ratio of firefly luciferase to Renilla luciferase. A p-value of less than 0.05 was assigned significance. Differences between group means were tested for statistical significance (p < 0.05) by appropriate post hoc tests.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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stimulation, we incubated cells overnight with different doses of TNF- (100, 10, 1 ng/mL) for 24 or 48 hrs. Interleukin-6 production by TNF- was stimulated in MG-63 cells, in a time- and dose-dependent manner, as measured by ELISA (Fig. 2A), while pre-incubation with the p38 MAPK inhibitor SB203580HCl caused a statistically significant decrease in TNF--induced IL-6 secretion (Fig. 2B). Western blotting confirmed that TNF- stimulation led to activation of the p38 MAPK pathway. Within 5 min, a rapid increase in levels of phosphorylated p38 MAPK was detected, after which levels gradually diminished (Fig. 3A). Additional experiments confirmed that SB203580 HCl inhibited phosphorylation of p38 MAPK (Fig. 3B).
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led to enhanced promoter activity in this model system, an effect that was reversed if cells were pre-incubated with SB203580 HCl (Fig. 4). Interestingly, the degree of inhibition of protein secretion was greater than that observed for gene expression. When two 5'-deletion constructs representing the proximal 490 bp and the proximal 260 bp of the IL-6 gene promoter were similarly stimulated, both promoter constructs responded similarly to the full-length promoter, indicating that elements responsible for TNF- stimulation and for p38 MAPK effects resided in the proximal 260-bp segment of the IL-6 gene promoter (Fig. 4A).
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B, we investigated whether TNF- stimulated the activity of either of these transcription factors as evidenced by promoter/reporter constructs specifically sensitive to the activation of C/EBPß or NFB. Similarly to previous reports, TNF- stimulated NFB activity in a manner dependent upon p38 MAPK phosphorylation and was p38-MAPK-dependent. In contrast, however, basal C/EBPß activity was inhibited by TNF-, an effect that was reversed by the inhibition of p38 MAPK phosphorylation (Fig. 4B
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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, is found in elevated concentrations in inflamed tissues adjacent to bone and induces IL-6 production by osteoblasts. The presence of IL-6 in the immediate vicinity of bone surfaces leads to the recruitment of osteoclast precursor cells (derived from monocyte cell lineage) as well as the initiation of differentiation of these precursor cells. Mature, multi-nucleated osteoclasts are, of course, the cell type responsible for the dissolution of bone. Therefore, by secreting IL-6 and predisposing to increased numbers of osteoclast precursor cells, osteoblasts are able to increase the rate of bone resorption at a given bone surface. Napoleon Bonaparte is reputed to have stated that "there are no bad armies, only bad generals". Therefore, to extend the analogy, osteoclasts constitute the army and osteoblasts act as the generals.
In preliminary experiments, using the MG-63 human osteosarcoma cell line, we showed that TNF- induces IL-6 secretion into culture supernatants by these cells. Using an inhibitor specific to p38 MAPK, we showed further that this induction is, at least in part, due to activation of the p38 MAPK pathway, as measured by phosphorylation of p38 MAPK by its activator, MKK6 (MAP Kinase Kinase 6). In higher eukaryotes, 4 p38 MAPKs have been identified to date: p38, p38ß/p38ß2, p38
/ERK6, and p38
/SAPK4. All of these proteins can be activated by MKK6, while only some can be activated by MKK3 and MKK4 (Alonso et al., 2000).
We further determined that the proximal 260-bp region of the IL-6 gene promoter was responsive to p38 MAPK activation. The downstream substrates of the p38 MAPK pathway include various protein kinases, including MAPK-APK-2 and PRAK, as well as various transcription factors, such as NFB, C/EBPs, MEF-2C, and ATF-2 (Schaeffer and Weber, 1999). Of these transcription factors, two have binding sites within the proximal 260 bp of the IL-6 gene promoter; specifically, an NFB binding site is located between positions -73 and -63, and a C/EBPß site is located between positions -173 and -145 relative to the transcription start site (Akira and Kishimoto, 1992). Although the NFB binding site has been suggested to be the principal regulatory site for IL-6 gene expression, the C/EBPß binding site also may influence the IL-6 gene response to pro-inflammatory cytokines (Merola et al., 1996; Galien and Garcia, 1997). Analysis of our data indicates that the deletion of a more distal AP-1 binding site (-283 to 277) does not alter the IL-6 gene promoter response to TNF-, confirming previous reports that the AP-1 binding site does not confer responsiveness to pro-inflammatory cytokines and is likely associated with the response to growth factors (Ray et al., 1989; Eickelberg et al., 1999; Franchimont et al., 1999). However, it appears that TNF- enhances functional activity of NFB while slightly diminishing the activity of C/EBPß relative to unstimulated cells. Of additional interest, both the NFB activation and the C/EBPß inhibition appear to be associated with activation of the p38 MAPK pathway, but to opposite effects. Whereas inhibition of the p38 MAPK pathway diminishes NFB activation, it prevents the TNF--induced inhibition of C/EBPß. However, it appears that, of the two effects, the activation of NFB is dominant, since the net effect of TNF- stimulation is enhanced IL-6 gene expression and enhanced IL-6 secretion. These findings are especially interesting in light of previous reports indicating that although the NFB binding site within the IL-6 gene promoter is necessary for lipopolysaccharide or TNF- responsiveness, its activity is dependent upon the presence of an intact C/EBPß binding site (Merola et al., 1996; Hu et al., 2000) and interaction between the two factors.
MG-63 osteosarcoma cells represent a transformed cell line and, in cell culture, initially resemble immature osteoblasts but can be modulated to express the phenotype of a mature osteoblast. Therefore, inasmuch as the data presented were obtained from a cell line, our results should be interpreted with appropriate caution. However, it is important to recognize that the p38 MAP kinase pathway, investigated here and by others (Chae et al., 2001) in MG-63 cells, has been shown to be activated by cytokines in vivo (Kumar et al., 2001) as well as in vitro by other osteoblast-like cell lines (Blanque et al., 1997; Kozawa et al., 1999; Miwa et al., 1999), and also in primary chondrocyte cultures (Kumar et al., 2001). In general, our findings corroborate the role of TNF- as a stimulator of p38 MAP kinase activity as reported by Kozawa et al. (1999) and by Miwa et al. (1999). That activated p38 MAP kinase activity leads to IL-6-induction-dependent NFB function has also been recently reported by Chae et al. (2001). Furthermore, we also show that p38 MAP kinase activity regulates the activity of a c/EBPß-responsive promoter in MG-63 cells. Previous reports have offered conflicting data in this regard. Caivano and Cohen (2000) noted that IL-1 induction of ERK1/2 and p38 MAP kinase had no effect on c/EBPß function in RAW264 macrophages, whereas Baldassare et al. (1999) demonstrated that p38 MAP kinase stimulation in response to lipopolysaccharide led to enhanced c/EBPß activity in RAW264.7 macrophages. However, neither of these reports focused on the role of p38 MAP kinase or c/EBPß on IL-6 production.
The therapeutic potential of modulating MAP kinase-dependent IL-6 production by osteoblastic cells has risen with recent findings by Badger et al. (2000) and by Kumar et al. (2001). The former group demonstrated that joint integrity in rats with adjuvant-induced arthritis could be significantly improved by treatment with the p38 MAP kinase inhibitor SB242235, as reflected by a variety of measures including inflammation and bone mineral density. Of specific interest, serum IL-6 levels were significantly reduced in rats treated with SB242235. More recently, Kumar et al. (2001) have reported that the induction of IL-6 production and bone resorption by IL-1 and TNF- in osteoblasts and chondrocytes could be inhibited by pre-treatment with SB203580. The authors conclude that there is a strong correlation between inhibition of p38 MAP kinase activity and inflammatory cytokine-stimulated biological responses as seen in joint diseases such as rheumatoid arthritis. These reports highlight the potential for alleviating the discomfort of bone/cartilage resorption-associated disease processes. However, it should be noted that IL-6 secretion was not completely inhibited in these studies or in those that we conducted, indicating that TNF- uses other mechanisms by which to stimulate IL-6 gene expression and/or secretion. The most likely mechanism is via stimulation of the ERK pathway, and we (data not shown) and others have shown that ERK activation by inflammatory cytokines will promote IL-6 secretion in a manner similar to that promoted by p38 MAP kinase (Vanden Berghe et al., 1998; Miwa et al., 1999). Furthermore, in our experiments, inhibition of p38 MAP kinase led to a greater inhibition of protein secretion than of gene expression. Since p38 MAP kinase activation has been reported also to play a role in translation/protein synthesis, it is possible that inhibition of transcription and translation events would manifest when IL-6 secretion was measured, whereas only effects on transcription would be observed when gene expression was assessed. However, further study is needed for definitive determination of the reasons for the differences in effects on IL-6 secretion and gene expression.
In conclusion, our results indicate that p38 MAP kinase activity is increased in MG-63 cells in response to stimulation with TNF-, and that the increased activity leads to enhanced IL-6 secretion, apparently as a result of enhanced NFB activity, despite the fact that TNF- may act to diminish the activity of C/EBPß. These results may aid us in the search for a better understanding of the cellular and molecular mechanisms involved in IL-6 production in osteoblasts. In turn, a better understanding of the cellular and molecular mechanisms of inflammatory bone resorption will lead to the development of effective therapeutic strategies to prevent or minimize inflammatory bone loss.
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ACKNOWLEDGMENTS |
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Received October 12, 2000; Last revision November 14, 2001; Accepted November 27, 2001
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| IADR Journals | Advances in Dental Research ® |
| Journal of Dental Research ® | Critical Reviews (1990-2004) |
