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J Dent Res 86(4):363-367, 2007
© 2007 International and American Associations for Dental Research

RESEARCH REPORT
Biological

Role of NF-{kappa}B in TNF-{alpha}-induced COX-2 Expression in Synovial Fibroblasts from Human TMJ

J. Ke1, X. Long1,2,*, Y. Liu3, Y.F. Zhang4, J. Li2, W. Fang2, and Q.G. Meng2

1 Key Lab. for Oral Biomedical Engineering, Ministry of Education,
2 Departments of Oral Maxillofacial Surgery and
4 Prosthodontics, School & Hospital of Stomatology,
3 Department of Radio-Chemotherapy of Zhongnan Hospital, Wuhan University, Wuhan 430079, PR China

* corresponding author, longxing_china{at}hotmail.com


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the temporomandibular joint (TMJ) synovium, cyclo-oxygenase-2 (COX-2) expression has been believed to be directly related to joint pain and synovitis. Here we investigated the role of Nuclear Factor {kappa}B (NF-{kappa}B) in the regulation of COX-2 expression in synovial fibroblasts from human TMJ induced by tumor necrosis factor-{alpha} (TNF-{alpha}). By reverse-transcriptase/polymerase chain-reaction (RT-PCR) and Western blotting analysis, TNF-{alpha} induced a dose- and time-dependent increase in COX-2 expression. Electrophoretic mobility shift assay (EMSA) revealed that transient NF-{kappa}B activation in the COX-2 promoter was triggered by TNF-{alpha}. In parallel with transient NF-{kappa}B activation, the rapid translocation of NF-{kappa}B, particularly the p65 subunit, from the cytoplasm into the nucleus was demonstrated. Pre-treatment with pyrolidine dithiocarbamate (PDTC), one of the NF-{kappa}B inhibitors, prevented binding to the COX-2 promoter and expression of COX-2 protein in response to TNF-{alpha}. These findings indicate that activation of NF-{kappa}B is responsible for TNF-{alpha}-induced COX-2 expression in synovial fibroblasts from the TMJ.

KEY WORDS: TMJ • COX-2 • NF-{kappa}B • TNF-{alpha} • synovial fibroblasts


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The molecular mechanism and the relative importance of specific factors for the athogenesis of TMJ disorders have not been well-characterized. Overproduction of inflammatory cytokines is believed to mediate the acute and chronic inflammation and associated connective tissue degeneration seen in TMJ disorders (Kacena et al., 2001). In particular, TNF-{alpha} appears to be the major proinflammatory cytokine involved in TMJ destruction. An elevated level of TNF-{alpha} in the synovial fluid has been detected in persons with TMJ disorder (Takahashi et al., 1998), and TNF-{alpha} stimulated chemokine expression in synovial fibroblasts from TMJ in vitro (Ogura et al., 2005). In addition, a transgenic mouse over-expressing TNF-{alpha} developed remarkable arthritic changes in the TMJ (Puzas et al., 2001).

Recent studies have demonstrated that prostaglandin E2 (PGE2) contributes to joint pain and bone absorption in TMJ (Alstergren and Kopp, 2000). PGE2 synthesis involves conversion of arachidonic acid by the cyclo-oxygenase (COX) enzymes to the general prostanoid precursor PGH2. COX has different isoforms, including constitutive COX-1 (and its splice variant, COX-3) and inducible COX-2 (Martel-Pelletier et al., 2003). Increased COX-2 expression has been detected in vivo by specific immunohistochemical analysis in the synovial tissue with internal derangement (Seki et al., 2004), and may be involved in angiogenesis of the synovial membrane and erosion of cartilage in inflamed joints (Myers et al., 2000).

The promoter region of the human COX-2 gene contains specific motifs with sequence similarity to the consensus binding site for NF-{kappa}B (Martel-Pelletier et al., 2003). NF-{kappa}B is composed of homo- and heterodimeric complexes of members of the Rel protein family, containing p65, p50, p52, c-Rel, and Rel B. NF-{kappa}B normally resides in the cytoplasm, where it is retained by association with I{kappa}B protein ({alpha}, ß, and {gamma}), an endogenous inhibitor. A variety of extracellular stimuli triggers the degradation of I{kappa}B by the proteasome pathway. Subsequently, NF-{kappa}B released from I{kappa}B translocates into the nucleus, binds to the regulatory element of the target genes, and controls their transcription (Barnes and Karin, 1997). The activated NF-{kappa}B plays an important role in regulating the expression of a variety of genes involved in immune and inflammatory responses (Ghosh et al., 1998).

The synovial membrane covers the inner face of the TMJ capsule and is populated by stromal cells and lining cells, which are fibroblasts. It has been accepted that these synovial fibroblasts play a key role in the process of TMJ disorders, based on their ability to degrade the extracellular matrix (Song and Windsor, 2005), and to provide chemotactic and active signals to infiltrating immunocytes (Ogura et al., 2005). However, whether TNF-{alpha} can induce COX-2 expression in these cells is still unknown. A likely signal transduction pathway for TNF-{alpha} involves the dephosphorylation of the inhibitor nuclear factor to form NF-{kappa}B (Fujisawa et al., 1996). In support of that, there are studies in intestinal epithelial cells indicating that COX-2 induction by TNF-{alpha} occurs through activation of NF-{kappa}B (Jobin et al., 1998). In contrast, recent studies by Chen et al.(1999) demonstrated that TNF-{alpha} induces increases in COX-2 expression in vascular smooth-muscle cells which occurred independently of NF-{kappa}B. Therefore, this study examined COX-2 expression in synovial fibroblasts from the TMJ, and investigated the role of NF-{kappa}B in the regulation of COX-2 expression in these cells with the treatment of TNF-{alpha}.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Synovial Fibroblast Culture
Synovial tissues were obtained from three patients (17, 28, and 60 yrs old) with condylar fracture undergoing arthrotomy treatment. The samples obtained from synovial regions without inflammation were verified based on a pathologic diagnosis. Informed consent was obtained, and the protocol was approved by the Human Research Ethics Committee, School & Hospital of Stomatology, Wuhan University. Synovial fibroblasts were isolated by enzymatic digestion of synovial tissues, as described previously (Alaaeddine et al., 1997). Synovial fibroblasts were grown in DMEM (Gibco, Grand Island, NY, USA), supplemented with 10% fetal calf serum, penicillin (100 unit/mL) and streptomycin (100 µ g/mL), in a humidified atmosphere of 5% CO2 in air. The cells between the fourth and eighth passages were used, during which time they constituted a homogenous population.

RNA Isolation and RT-PCR
Total RNA was isolated from cells with the use of Trizol reagent (Invitrogen, Carlsbad, CA, USA) and quantified by spectrophotometry (Shimadzu Corporation, Tokyo, Japan). First-strand cDNA was synthesized from 1 µg of total RNA via the SuperscriptTM reverse-transcription system (TaKaRa Biotechnology Co., Dalian, China) with oligdT primer. PCR products were analyzed by electrophoresis in 1.5% agarose gel containing ethidium bromide (see APPENDIX).

Whole-cell, Cytoplasmic, and Nuclear Extract Preparation
Whole-cell proteins were harvested by the methods of Li et al.(1994), and nuclear extracts were prepared according to a protocol modified from the method described by Schreiber et al.(1989). Protein concentrations were determined by use of the Bicinchoninic acid protein assay kit (Pierce Chemical Company, Rockford, IL, USA) (see APPENDIX).

Western Blotting Analysis
Samples (80 µg of whole-cell lysates or 50 µg protein each from cytoplasmic and nuclear extracts) underwent electrophoresis in 10% sodium dodecyl sulfate polyacrylamide gels, and were transferred to polyvinylidene difluoride membranes. Bound antibodies were detected with 3,3'-Diaminobenzidine (see APPENDIX).

EMSA
We performed NF-{kappa}B binding by incubating 5 µg of nuclear extract in 10 µL of binding buffer, containing 1 ng of the 32P-labeled DNA probe (40,000 cpm) and 1 µg of poly(dI-dC), for 30 min at room temperature. A 100-fold excess of the unlabeled oligonucleotide was used for competition. For supershift assay, polyclonal antibodies specific for p65 and p50 were added to the binding reaction prior to addition of the labeled probes, and were incubated on ice for 3 hrs. The DNA-protein complex was analyzed on native 5% polyacrylamide gels in TBE buffer. The gels were then dried and subjected to autoradiography (see APPENDIX).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of TNF-{alpha}-induced COX-2 Expression in Synovial Fibroblasts from the TMJ
Cells were exposed to increasing concentrations of TNF-{alpha} (from 1 to 40 ng/mL) during 2 or 6 hrs, and the levels of COX-2 mRNA and protein were subsequently evaluated. The incubation with TNF-{alpha} resulted in a dose-dependent increase in the levels of COX-2 mRNA (Fig. 1AGo). The induction of COX-2 protein was perceptible following stimulation with 10 ng/mL of TNF-{alpha}, and thereafter followed a pattern of biosynthesis closely resembling COX-2 mRNA expression (Fig. 1BGo).


Figure 1
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  		Figure 1. Dose-dependent TNF-{alpha}-induced COX-2 mRNA and protein in synovial fibroblasts from TMJ. (A) Cells were incubated with various concentrations of TNF-{alpha} for 2 hrs. Total RNA was isolated from cells and analyzed for COX-2 and GAPDH mRNA by RT-PCR. (B) Cells were incubated with various concentrations of TNF-{alpha} for 6 hrs. Whole-cell lysates were prepared and subjected to Western blotting with antibody specific for COX-2 and actin. The data represent 1 of 3 separate experiments with similar results.

 
Also, exposure of synovial fibroblasts from TMJ to TNF-{alpha} (20 ng/mL) resulted in a time-dependent increase in COX-2 expression (Figs. 2A, 2BGo). An increase in COX-2 mRNA expression was noted after cells were treated for 0.5 hrs with TNF-{alpha}, and reached a peak at 2 hrs, and declining gradually thereafter to baseline, after 12 hrs (Fig. 2AGo). Parallel cultures maintained without TNF-{alpha} treatment for identical periods of time failed to demonstrate significant alterations in the expression of the COX-2 gene, although a constitutive expression was observed (Fig. 2AGo). The expression of COX-2 protein was detectable at 6 hrs following stimulation with TNF-{alpha} , and gradually increased over time (Fig. 2BGo).


Figure 2
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  		Figure 2. Time-dependent TNF-{alpha}-induced COX-2 mRNA and protein in synovial fibroblasts from TMJ. (A) Cells were incubated with 20 ng/mL of TNF-{alpha} for various time intervals. Total RNA was then isolated from cells and analyzed for COX-2 and GAPDH mRNA by RT-PCR. (B) Cells were incubated with 20 ng/mL of TNF-{alpha} for various time intervals, and the whole-cell lysates were prepared and subjected to Western blotting with antibody specific for COX-2 and actin. The data represent 1 of 3 separate experiments with similar results.

 
Characterization of TNF-{alpha}-induced Activation of NF-{kappa}B
The time-course of NF-{kappa}B activation after treatment with TNF- {alpha} (20 ng/mL) was examined by EMSA. In the presence of TNF-{kappa}, rapid NF-{kappa}B activation was observed as early as 20 min, peaked at 40 min, and declined to weak status after 120 min (Fig. 3AGo). In untreated cells, no NF-{kappa}B-specific DNA-protein complex was identified (Fig. 3AGo).


Figure 3
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  		Figure 3. Time-course of TNF-{alpha}-induced NF-{kappa}B-specific DNA-protein complex formation and NF-{kappa}B translocation in synovial fibroblasts from TMJ. Cells were treated with 20 ng/mL TNF-{alpha} for various time intervals, and then cytoplasmic and nuclear extracts were prepared. (A) NF-{kappa}B-specific DNA-protein-binding activity in nuclear extracts was determined by EMSA. (B) Supershift assays were performed with specific p65 and p50 antibodies. Moreover, we added unlabeled NF-{kappa}B probes in a 100-fold excess, to determine the specificity of NF-{kappa}B-specific DNA protein. (C) Cytoplasmic and nuclear levels of p65 and p50 proteins were detected by Western blotting. The data represent 1 of 3 separate experiments with similar results.

 
To identify the specific subunits involved in the formation of the banding pattern of the NF-{kappa}B dimer after TNF-{alpha} stimulation, we performed supershift assays with antibodies to p50 and p65. Incubation with anti-p50 or anti-p65 antibodies induced a supershift (Fig. 3BGo). The addition of a 100-fold excess of unlabeled NF-{kappa}B caused the band to disappear completely.

TNF-{alpha}-induced Translocation of p65 from the Cytoplasm to the Nucleus
Nuclear and cytoplasmic extracts from synovial fibroblasts from TMJ stimulated with TNF-{alpha} for the various times were examined by Western blot analysis. Very little p65 was evident in the nuclei of the untreated cells (Fig. 3CGo). After 20 min of TNF-{alpha} induction, nuclear p65 began to appear, peaking at 40 min and declining again by 120 min (Fig. 3CGo). A concomitant decrease in cytoplasmic p65 corresponded to the observed increase in nuclear p65 (Fig. 3CGo).

In the case of p50, again, little protein was evident in the nuclei of untreated cells (Fig. 3CGo). However, unlike p65, the level of nuclear p50 seemed relatively constant over 120 min, with only a slight increase observed at 40 min (Fig. 3CGo).

PDTC Inhibited TNF-{alpha}-induced NF-{kappa}B Activation and COX-2 Expression
Synovial fibroblasts from TMJ were pre-treated with the NF-{kappa}B inhibitor PDTC (50 µmol/L) for 1 hr and then incubated with TNF-{alpha} (20 ng/mL). TNF-{alpha}-dependent activation of NF-{kappa}B was inhibited by PDTC (Fig. 4AGo). PDTC also inhibited TNF-{alpha}-induced COX-2 protein levels (Fig. 4BGo). PDTC alone did not exert a significantly cytotoxic effect at the concentration applied, as examined by MTT assays (data not shown).


Figure 4
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  		Figure 4. Inhibitory effect of PDTC on NF-{kappa}B activity and COX-2 protein expression induced by TNF-{alpha} in synovial fibroblasts from the TMJ. (A) NF-{kappa} B binding activity in the nuclear extracts from cells pre-treated with PDTC (50 µmol/L) 1 hr before 20 ng/mL of TNF-{alpha} stimulation for 40 min was determined by EMSA. (B) COX-2 protein expression in the whole-cell lysates from cells pre-treated with PDTC (50 µmol/L) 1 hr before 20 ng/mL of TNF-{alpha} stimulation for 6 hrs was assayed by Western blotting. The data represent 1 of 3 separate experiments with similar results.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the TMJ synovium, enhanced COX-2 expression has been believed to be directly related to joint pain and synovitis (Seki et al., 2004), although the factors involved in the regulation of COX-2 expression in TMJ have not been well-characterized. We have previously reported that hypoxia stimulates COX-2 gene and protein expression in synovial fibroblasts from the TMJ (Ke et al., 2005). In this study, we demonstrated that TNF-{alpha} induced a dose- and time-dependent increase in COX-2 expression in these cells. Similar observations were published by Chin et al.(2001) with respect to COX-2 expression in response to interleukin-1ß (IL-1ß) and TNF-{alpha} in human retinal pigment epithelial cells. The inducible COX-2 enzyme is responsible for the prompt increase of PGE2 during the inflammatory response. PGE2 stimulates vasodilation, increases vascular permeability, and contributes to nociceptive sensitization in the inflamed tissue (Vane et al., 1994). Therefore, our results may partially explain the fact that the enhanced TNF-{alpha} in the synovial fluid of TMJ disorders is associated with local joint pain (Takahashi et al., 1998).

However, the cellular mechanism for the increased expression of COX-2 following TNF-{alpha} treatment remains unclear. Thus, we initially focused on the question whether TNF-{alpha} treatment triggers activation of NF-{kappa}B in the COX-2 promoter in synovial fibroblasts from the TMJ. There are two NF-{kappa}B consensus sites in the promoter region of the human COX-2 gene, the NF-{kappa}B-5' site (–447/–438) and the NF-{kappa}B-3' site (–223/–214) (Tazawa et al., 1994). The NF-{kappa}B-5' site has been shown to be involved in TNF-{alpha}-induced COX-2 expression in a murine osteoblast cell line (Yamamoto et al., 1995), while the NF-{kappa}B-3' site, in concert with the nuclear factor-interleukin-6 (NF-IL6) and cyclic adenosine monophosphate response element (CRE) sites, may play a role in facilitating the induction of COX-2 by lipopolysaccharide (LPS) and phorbol ester in bovine aortic endothelial cells (Inoue et al., 1995). Thus, in the present study, the NF-{kappa}B-5' site was used as the sequence of the DNA binding probe. Analysis of our data showed the transient NF-{kappa}B activation in the COX-2 promoter with the treatment of TNF-{alpha}. Moreover, the supershift assay confirmed the specificity of the NF-{kappa}B DNA-protein complex, and demonstrated the presence of the p65/p50 NF-{kappa}B heterodimer in synovial fibroblasts from TMJ exposed to TNF-{alpha}. These in vitro results are consistent with previous findings that an active form of NF-{kappa}B (p65/p50) was expressed in rat TMJ synovium in vivo, after induced synovitis (Yamaza et al., 2003).

In parallel with transient NF-{kappa}B activation in the COX-2 promoter, the rapid translocation of NF-{kappa}B from the cytoplasm into the nucleus in synovial fibroblasts from the TMJ was demonstrated by Western blotting analysis. Our results clearly exhibited that TNF-{alpha} induced an early and rapid nuclear translocation of p65, while p50 appeared to be relatively unresponsive. These findings agree with those of Marok et al.(1996), who reported that the immunoreactive p65 was predominantly situated in the nuclei of the synovial lining cells from persons with RA. Therefore, nuclear translocation of NF-{kappa}B p65 in synovial fibroblasts, not p50, seems to be the critical step during inflammation in the synovium.

The activation of NF-{kappa}B can be blocked by PDTC, which leaves the DNA binding activity of other transcription factors unaffected (Schreck et al., 1992). PDTC has been shown to prevent the induction of COX-2 in TNF-{alpha}-stimulated human tracheal smooth-muscle cells (Lin et al., 2004). Alternatively, Takano et al.(2001) reported that PDTC inhibited complement C5b-9-mediated nuclear translocation of NF-{kappa}B, but failed to inhibit the expression of COX-2. They concluded that, in rat glomerular epithelial cells, complement C5b-9-stimulated COX-2 expression is activated through a transcription factor different from NF-{kappa}B. In the present study, PDTC prevented binding to the COX-2 promoter, and the expression of COX-2 protein in synovial fibroblasts from the TMJ in response to TNF-{alpha}. These findings further confirmed the results of our EMSA experiments, and indicated that NF-{kappa}B is essential for COX-2 induction in response to TNF-{alpha} in synovial fibroblasts from TMJ.

Interestingly, NF-{kappa}B may not be the only transcription factor involved in the TNF-{alpha}-induced up-regulation of the COX-2 gene in synovial fibroblasts, because PDTC treatment of these cells incubated with TNF-{alpha} did not absolutely inhibit production of COX-2. Recently, several other transcription factors have been demonstrated to regulate COX-2 gene expression in response to TNF-{alpha} in various cells. Transcription of COX-2 in mouse MC3T3-E1 cells requires both NF-{kappa} B and NF-IL6 activation (Yamamoto et al., 1995), with constructs containing the mouse COX-2 gene promoter linked to a luciferase reporter gene. Schroer et al.(2002) reported that transient transfection of a specific COX-2 promoter fragment bearing the CRE mutation decreased COX-2 promoter activity induced by TNF-{alpha}, and they suggested that CRE is necessary for COX-2 gene expression in human umbilical vein endothelial cells. Thus, in TNF-{alpha}-stimulated synovial fibroblasts from the TMJ, NF-IL6 and CRE involvement in transcriptional regulation of COX-2 remains to be evaluated.

In conclusion, this study demonstrated that the activation of NF-{kappa}B is responsible for TNF-{alpha}-induced COX-2 expression in synovial fibroblasts from the TMJ. These results may be helpful to our understanding of the molecular mechanisms underlying increased COX-2 expression and, indirectly, PGE2 production associated with elevated levels of TNF-{alpha} in synovial tissues and fluid from persons with TMJ disorder.


   ACKNOWLEDGMENTS
 
This study was supported by a grant (No. 30371549) from the National Science Foundation of China.


   FOOTNOTES
 
A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org.

Received March 28, 2006; Last revision November 30, 2006; Accepted December 5, 2006


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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