Kikumori T, Kambe F, Nagaya T, Imai T, Funahashi H, Seo H. 1998 Activation of transcriptionally active nuclear factor-. kappaB by tumor necrosis factor-alpha ...
JOURNAL OF BONE AND MINERAL RESEARCH Volume 16, Number 7, 2001 © 2001 American Society for Bone and Mineral Research
Tumor Necrosis Factor ␣ Induces Expression of Genes for Matrix Degradation in Human Chondrocyte-like HCS-2/8 Cells Through Activation of NF-B: Abrogation of the Tumor Necrosis Factor ␣ Effect by Proteasome Inhibitors TADAHIRO SAKAI,1,2 FUKUSHI KAMBE,1 HIROHITO MITSUYAMA,1,2 NAOKI ISHIGURO,2 KAZUTOSHI KUROKOUCHI,2 MASAHARU TAKIGAWA,3 HISASHI IWATA,2 and HISAO SEO1
ABSTRACT Tumor necrosis factor ␣ (TNF-␣) has been suggested to induce chondrocytic chondrolysis in both inflammatory and degenerative joint diseases. However, its intracellular signaling pathway leading to the chondrolysis has not been studied in detail. Thus, we investigated whether TNF-␣ activates a transcription factor nuclear factor B (NF-B) in human chondrocyte-like cells (HCS-2/8) and induces the expression of genes involved in the degradation of cartilage matrix. Treatment of the cells with TNF-␣ markedly increased the levels of matrix metalloproteinase 1 (MMP-1), MMP-3, intercellular adhesion molecule 1 (ICAM-1), and cyclo-oxygenase 2 (COX-2) messenger RNAs (mRNAs). The increase in the mRNAs was associated with the activation of p65/p50 heterodimer NF-B. IB-␣ and IB-, cytoplasmic molecules preventing the nuclear translocation of NF-B, were degraded rapidly by TNF-␣ followed by their synthesis to the basal level. Treatment with proteasome inhibitors inhibited the degradation of both IB-␣ and IB- and prevented the TNF-␣– dependent nuclear translocation of p65. Furthermore, the inhibitors completely prevented the TNF-␣– dependent induction of MMP-1, MMP-3, ICAM-1, and COX-2 mRNAs. Thus, it is shown that the activation of p65/p50 NF-B by TNF-␣ plays a cardinal role in inducing the expression of MMP-1, MMP-3, ICAM-1, and COX-2 genes, which are involved in matrix degradation and inflammatory reaction in chondrocytes, leading to chondrocytic chondrolysis. (J Bone Miner Res 2001;16:1272–1280) Key words:
chondrocytes, tumor necrosis factor ␣, nuclear factor B, IB, proteasome inhibitors
INTRODUCTION (1) HONDROCYTE IS the only cell type in articular cartilage and plays a pivotal role in its remodeling and maintenance by controlling synthesis and degradation of the matrix.(2) In both inflammatory and degenerative joint diseases, a cytokine tumor necrosis factor ␣ (TNF-␣) produced from chondrocytes or infiltrating cells has been suggested to
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induce the chondrocytic chondrolysis. In osteoarthritis (OA), the number of chondrocytes that synthesize TNF-␣ have been shown to increase in OA cartilage.(3,4) Rheumatoid arthritis (RA) often is associated with destruction of articular cartilage and the inflammatory sites were infiltrated with neutrophils and monocytes/macrophages that produce TNF-␣.(4 – 6) Studies using cultured chondrocytes or chondrocyte-like cell lines have shown the existence of functional TNF-␣
1 Department of Endocrinology and Metabolism, Division of Molecular and Cellular Adaptation, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan. 2 Department of Orthopedics, Nagoya University School of Medicine, Nagoya, Japan. 3 Department of Biochemistry, Molecular Dentistry, Okayama University Dental School, Okayama, Japan.
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receptor(7,8) and pleiotropic effects of TNF-␣ on the cells. For example, TNF-␣ has been shown to stimulate the synthesis of matrix metalloproteinase 1 (MMP-1; collagenase1), MMP-3 (stromelysin 1),(9 –11) adhesion molecules such as intercellular adhesion molecule 1 (ICAM-1),(12) and cyclo-oxygenase 2 (COX-2).(13) However, the intracellular signaling pathway of TNF-␣ in chondrocytes has not been studied in detail. In various cell types, TNF-␣ exerts its action by binding to a membrane receptor with subsequent activation of a dimeric transcription factor nuclear factor B (NF-B) consisting of the Rel family proteins such as p65 (RelA), p50, p52, c-Rel, and RelB.(14) In unstimulated cells, NF-B is present in the cytoplasm and is bound to IB that prevents the NF-B from entering the nucleus. Several IB isoforms such as IB-␣ and IB- have been identified.(15,16) Stimulation of TNF-␣ activates IB kinases, which specifically phosphorylate IB, resulting in IB ubiquitination and subsequent degradation of IB by the ubiquitin-proteasome pathway. Thus, NF-B released from IB translocates into the nucleus, binds to the regulatory element of the target genes, and controls their transcription.(17,18) In this study, we used human chondrocyte-like cells (HCS-2/8), which maintain the phenotype of chondrocytes; they produce cartilage-specific proteoglycans and synthesize the type II collagen but not type I collagen(19) to investigate a possible involvement of NF-B in TNF-␣– dependent expressions of MMP-1, MMP-3, ICAM-1, and COX-2. It will be shown that p65/p50 heterodimer NF-B is activated by TNF-␣ with concomitant degradation of both IB-␣ and IB-. Furthermore, the use of proteasome inhibitors will show that TNF-␣– dependent induction of MMP-1, MMP-3, ICAM-1, and COX-2 genes is regulated by NF-B.
MATERIALS AND METHODS Cell culture HCS-2/8 cells, a chondrocyte cell line established from a human chondrosarcoma,(19) were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum. The nearly confluent cells in a 21-cm2 culture dish (Falcon 3002; Becton Dickinson & Co., Lincoln Park, NJ, USA) were incubated with recombinant human TNF-␣ (2.5 ⫻ 103 U/g; Asahi Chemical Industry Co., Ltd., Tokyo, Japan) for various lengths of time. Proteasome inhibitors, proteasome inhibitor I [PSI; Cbz-ile-glu (O-t-Bu)-ala-leucinal], MG132 (Cbz-leu-leu-leucinal), and Lactacystin were purchased from Calbiochem (San Diego, CA, USA). PSI, MG132, and Lactacystin were dissolved in dimethylsulfoxide (DMSO) at concentrations of 60, 50, and 10 mM, respectively. In experiments using these inhibitors, they were added to the cells at 1:1000 dilution 1 h before the addition of TNF-␣. Control cells were added with the vehicle DMSO at final concentration 0.1%. These concentrations of the inhibitors were shown to prevent the IB degradation in various cell types.(20 –22) After 6-h incubation with TNF-␣, the cells were harvested, and total RNA was extracted for Northern blot analysis by the acid guanidium
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thiocyanate-phenol-chloroform-extraction method.(23) For electrophoretic mobility shift assay (EMSA) and Western blot analysis, nuclear and cytosolic extracts were prepared as described previously.(24) Protein concentration of the extracts was determined by a microassay kit (Bio-Rad, Richmond, CA, USA) using bovine serum albumin (BSA) as a standard. In an experiment using epidermal growth factor (EGF), human recombinant EGF (Sigma Chemical Co., St. Louis, MO, USA) was added at the final concentration of 10 ng/ml.
Northern blot analysis The detailed procedure was described previously.(25) After fractionation of 20 g total RNA on 0.8% agarose gel, it was transferred onto a Gene Screen Plus membrane (New England Nuclear, Boston, MA, USA). The membrane was hybridized with human MMP-1, MMP-3, ICAM-1, COX-2, and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) complementary DNA (cDNA) probes labeled with [32P]deoxycytidine 5⬘-triphosphate (dCTP). After hybridization, the membrane was subjected to quantitative analysis using The BAS 2000 bioimage analyzing system (Fuji Film Co., Tokyo, Japan) and the levels of MMP-1, MMP-3, ICAM-1, and COX-2 messenger RNAs (mRNAs) were corrected by those of GAPDH mRNA. The membrane was then exposed to X-ray film for autoradiography. COX-2 cDNA was purchased from Cayman Chemical Co., (Ann Arbor, MI, USA). The cDNA for human ICAM-1 was cloned from TNF-␣–treated human osteosarcoma cell line (HOS-TE85) by reverse-transcription polymerase chain reaction (RT-PCR) using the following primers: 5⬘CATCTGTGTCCCCCTCAAAAG-3⬘ and 5⬘-TCGTTGCCATAGGTGACTGTG-3⬘. The amplified PCR product was ligated into pGEM-T-easy plasmid (TA cloning system; Promega, Madison, WI, USA). The authenticity of cDNA was verified by DNA sequencing. The preparation of human pro-MMP-1,(26) pro-MMP-3,(27) and GAPDH cDNAs was described in our previous reports.(28)
EMSA Nuclear extracts (10 g of protein) were used for EMSA. The detailed procedures of EMSA were described in our previous report.(29) The Bwt oligonucleotides (5⬘-TCGAGCAGAGGGGACTTTCCGAGAG-3⬘ and 5⬘-TCGACTCTCGGAAAGTCCCCTCTGC-3⬘) containing the canonical NF-B binding site (italic) identified in mouse immunoglobulin (Ig) enhancer(30) were annealed, labeled by Klenow enzyme in the presence of [32P]dCTP, and used as a probe. To identify the NF-B subunits, supershift analyses were performed using antibodies directed against p50, p52, p65, c-Rel, Rel B (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), and preimmune rabbit serum. They were added to the binding reaction mixture before the addition of the labeled probe and incubated for 1 h at 4°C. Samples were analyzed on 4% polyacrylamide gel (29:1, acrylamide/ bisacrylamide) containing 45 mM Tris-HCl (pH 8.0), 45 mM boric acid, and 1 mM EDTA (pH 8.0) for 4 h at 160 V
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FIG. 1. TNF-␣ increases MMP-1, MMP-3, ICAM-1, and COX-2 mRNA levels in a dosedependent manner in HCS-2/8 cells. The cells were incubated with various doses of TNF-␣ (0, 0.5, 5, and 50 U/ml) for 6 h. Total RNA was extracted and subjected to Northern blot analysis using MMP-1, MMP-3, ICAM-1, COX-2, and GAPDH cDNAs as probes. After the radioactivities of the bands were measured by the BAS 2000 system, the mRNA levels were normalized by those of GAPDH mRNA and then expressed as percentage of the level treated with TNF-␣ (50 U/ml). The data are expressed as mean ⫾ SD (n ⫽ 3). *p ⬍ 0.05 versus the level without TNF-␣. Representative autoradiographs are shown. A similar result was obtained from a separate experiment.
at 25°C. The gels were dried and autoradiographed with an intensifying screen at ⫺80°C.
Western blot analysis The detailed procedure was described previously.(31) In brief, cytosolic extracts (40 g of protein) and standards for molecular weight were fractionated on a sodium dodecyl sulfate (SDS)–10% polyacrylamide gel and electroblotted onto a Hybond-C super membrane (Amersham Corp., Arlington Heights, IL, USA). A part of the membrane with molecular weight marker was separated and stained with Coomassie Brilliant Blue R (Sigma Chemical Co.). The remaining membrane was soaked overnight in a blocking buffer (phosphate-buffered saline [PBS] containing 5% low-fat dried milk powder [Snowbrand Milk Products Co., Ltd., Tokyo, Japan]). Then, the membrane was incubated for 1 h with an antibody against either IB-␣ or IB- (Santa Cruz Biotechnology, Inc.) diluted at 1:1000 with the blocking buffer. After washing three times with PBS containing 0.1% Tween 20, the membrane was incubated for 1 h with anti-rabbit-IgG goat IgG conjugated with alkaline phosphatase (Zymed, San Francisco, CA, USA) diluted at 1:2000 with PBS containing 0.1% Tween 20. After washing three times, it was incubated in a color development solution containing nitro blue tetrazolium and 5-bromo-4chloro-3-indolyl-phosphate (NBT/BCIP Tablet; Boehringer Mannheim, Mannheim, Germany).
Immunocytochemistry HCS-2/8 cells were cultured in chamber slides (Lab-Tek chamber slides; Nalge Nunc International, Naperville, IL, USA). The detailed procedures were described previously.(32) The cells were treated with proteasome inhibitors for 1 h followed by TNF-␣ (50 U/ml) for 1 h. They were then fixed
in 3.75% paraformaldehyde in PBS for 25 minutes and permeabilized with 0.2% Triton X- 100 in PBS for 5 minutes. After a 15-minute incubation with 1% BSA in PBS, the cells were incubated with anti-p65 antibody (1:400 dilution; Santa Cruz Biotechnology, Inc.) overnight at 4°C. They were then washed in PBS and incubated with secondary antibody conjugated with fluorescein isothiocyanate (FITC; 1:100 dilution; Zymed) for 2 h. The cells were visualized with a confocal laser scanning microscope (LSM 510; Carl Zeiss, Jena, Germany).
Statistical analyses The results were expressed as mean ⫾ SD. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni test. A value of p ⬍ 0.05 was considered significant.
RESULTS TNF-␣ induces expression of MMP-1, MMP-3, ICAM1, and COX-2 HCS-2/8 cells were incubated with various concentrations of TNF-␣ for 6 h, and the expression of MMP-1, MMP-3, ICAM-1, COX-2, and GAPDH was determined by Northern blot analysis. As shown in Fig. 1, the basal expressions of MMP-1, MMP-3, ICAM-1, and COX-2 mRNAs were hardly detected. TNF-␣, at a final concentration of 5 U/ml, induced a marked increase in all the mRNAs. A single band for each mRNA was detected; the size of each mRNA was approximately 2.0 kilobases (kb) for MMP-1, 1.9 kb for MMP-3, 3.5 kb for ICAM-1, 4.4 kb for COX-2, and 1.3 kb for GAPDH. Increase in the dosage of TNF-␣ (50 U/ml) resulted in further increases in the mRNA levels.
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FIG. 2. Time course of TNF-␣– dependent induction of MMP-1, MMP-3, ICAM-1, and COX-2 mRNAs. HCS-2/8 cells were treated with TNF-␣ (50 U/ml) for various lengths of time. Northern blot analysis was performed. The levels of each mRNA were normalized by the GAPDH mRNA levels and then expressed as percentage of the maximum level after TNF-␣ stimulation. Similar result was obtained from a separate experiment. The data are expressed as mean ⫾ SD (n ⫽ 4). *p ⬍ 0.05 versus the level before TNF-␣.
Note that the levels of GAPDH mRNA were not altered by TNF-␣ at any concentrations. Time course of the induction by TNF-␣ (50 U/ml) is shown in Fig. 2. The levels of MMP-1, MMP-3, and ICAM-1 mRNAs significantly increased 3 h after TNF-␣ treatment and increased further until 12 h. However, the COX-2 mRNA level significantly increased within 1 h and reached a peak at 6 h. GAPDH mRNA levels were not affected by TNF-␣ at any time point. These results showed that TNF-␣ induces the expressions of MMP-1, MMP-3, ICAM-1, and COX-2 mRNAs in a dose- and timedependent manner in chondrocyte-like HCS-2/8 cells.
TNF-␣ induces activation of NF-B As shown in Fig. 3A, HCS-2/8 cells were treated with various concentrations of TNF-␣ for 1 h, and their nuclear extracts were subjected to EMSA. The binding activity of NF-B was not detected before stimulation with TNF-␣. After the treatment, it markedly induced the binding activity of NF-B in a dose-dependent manner. To characterize NF-B subunits, the nuclear extracts were incubated with specific antibodies raised against each subunit of NF-B and subjected to EMSA (Fig. 3B). Antip50 antibody slowed the migration of the NF-B/DNA complex (lane 3), and anti-p65 antibody supershifted the complex (lane 5). Other antibodies and preimmune normal rabbit serum did not affect the mobility of the complex, indicating that NF-B induced by TNF-␣ in HCS-2/8 cells consists of p65/p50 heterodimer. As shown in Fig. 3C, we examined time course of NF-B activation by TNF-␣ (50 U/ml). It rapidly induced the activation within 15 minutes, and the maximum DNA bind-
ing was observed at 3 h poststimulation. Thereafter, the DNA binding was reduced gradually until 12 h. To study how the activation of NF-B was associated with the degradation of IBs, Western blot analysis was performed by using cytosol extracts. As shown in Fig. 4, a single band of nonphosphorylated IB-␣ (35 kDa) was detected before TNF-␣ (time 0). At 5 minutes after its addition, an additional slower-migrating band representing phosphorylated IB-␣ appeared, followed by the disappearance of both bands from 15 to 30 minutes poststimulation. Then IB-␣ without phosphorylation gradually increased until 12 h. Interestingly, the time course of IB- degradation was different from that of IB-␣. Degradation of IB- (49 kDa) was noted at 30 minutes after TNF-␣ and lasted until 3 h, followed by the appearance until 12 h. It is noted that phosphorylated IB- was not detected.
Proteasome inhibitors attenuate TNF-␣– dependent activation of NF-B To examine whether NF-B mediates the TNF-␣– dependent induction of MMP-1, MMP-3, ICAM-1, and COX-2 mRNAs, we used proteasome inhibitors, which have been shown to prevent IB degradation and thereby NF-B activation.(20) To study the effect of inhibitors on the degradation of IB-␣, cytosol extract was prepared 15 minutes after TNF-␣, because degradation was evident at this time point (Fig. 4). As shown in Fig. 5A, the degradation of IB-␣ was inhibited completely by the proteasome inhibitors PSI, MG132, and Lactacystin. It was noted that both phosphorylated and nonphosphorylated forms of IB-␣ were prevented from degradation. The effect on IB-
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FIG. 3. TNF-␣ induces the activation of NF-B in HCS-2/8 cells. (A) The cells were treated with various doses of TNF-␣ (0, 0.5, 5, and 50 U/ml) for 1 h. Nuclear extracts (10 g/lane) were subjected to EMSA using the labeled oligonucleotide Bwt as a probe. (B) Nuclear extracts prepared from the cells treated with TNF-␣ (50 U/ml) for 1 h were subjected to supershift analysis using normal rabbit serum (NRS, lane 2) and antibodies directed against p50, p52, p65, c-Rel, and Rel B (lanes 3–7, respectively). The closed and open arrowheads indicate the NF-B/Bwt complex with anti-50 antibody and that with anti-p65 antibody, respectively. (C) The cells were treated with TNF-␣ (50 U/ml) for various lengths of time. Nuclear extracts (10 g/lane) were subjected to EMSA.
in the cytoplasm before TNF-␣. It induced translocation of p65 into the nucleus. Pretreatment with Lactacystin inhibited the translocation. Thus, it is shown that the proteasome inhibitors prevent the degradation of IBs induced by TNF-␣ and thereby inhibit TNF-␣– dependent activation NF-B in HCS-2/8 cells.
Proteasome inhibitors abrogate TNF-␣– dependent expression of MMP-1, MMP-3, ICAM-1, and COX-2
FIG. 4. Time course of degradation of cytoplasmic IB-␣ and IB- after TNF-␣. Cytosol extracts (40 g/lane) prepared from the cells treated with TNF-␣ (50 U/ml) for various lengths of time were subjected to Western blot analysis using anti-IB-␣ and IB- antibodies as the first antibodies.
degradation was studied at 60 minutes after TNF-␣, because it occurred later than that of IB-␣ (Fig. 4). The degradation of IB- also was inhibited by the proteasome inhibitors. Next, we studied effects of the proteasome inhibitors on the TNF-␣– dependent activation of NF-B. The cells were pretreated with each inhibitor for 1 h and then incubated with TNF-␣ together with the inhibitor for 1 h. As shown in Fig. 5B, pretreatment with the inhibitors markedly reduced the activation of NF-B. The effect of one of the inhibitors Lactacystin on the nuclear translocation of p65 was examined by immunocytochemical analysis. As shown in Fig. 6, p65 was detected
We then examined effect of the proteasome inhibitors on TNF-␣– dependent expression of MMP-1, MMP-3, ICAM-1, and COX-2 mRNAs (Fig. 7). After 1-h preincubation of HCS-2/8 cells with PSI, MG132, or Lactacystin, the cells were incubated with TNF-␣ for an additional 6 h. Induction of all the four mRNA levels was suppressed by the proteasome inhibitors. In contrast, GAPDH mRNA levels were not altered by the inhibitors. As shown in Fig. 8, the effect of the proteasome inhibitor MG132 was dose dependent. To evaluate the specificity of proteasome inhibitors on NF-B activation, we examined the effect of the inhibitors on the activator protein (AP)-1– dependent expression of MMP-1 by EGF.(9,33,34) As shown in Fig. 9, EGF increased the MMP-1 mRNA level. However, this increase was not prevented by the preincubation with PSI, MG132, or Lactacystin.
DISCUSSION This study showed that TNF-␣ induced a transient activation of the p65/p50 heterodimer NF-B (a peak at 3h) in
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FIG. 5. Effects of proteasome inhibitors on TNF-␣– dependent activation of NF-B and degradation of IB-␣ and IB-. (A) After 1-h preincubation of HCS-2/8 cells with PSI (60 M), MG132 (50 M), Lactacystin (10 M), or DMSO (0.1%), the cells were incubated with TNF-␣ (50 U/ml) for 15 minutes for the detection of IB-␣ and 60 minutes for IB-. Cytosol extracts were subjected to Western blot analysis using anti-IB-␣ and IB- antibodies as the first antibodies. The positions of IB-␣ and its phosphorylated form are indicated by closed and open arrowheads, respectively. (B) After preincubation with the proteasome inhibitors as described in panel A, the cells were incubated with TNF-␣ (50 U/ml) for 1 h. Nuclear extracts were subjected to EMSA using the labeled oligonucleotide Bwt as a probe. The representative autoradiograph of EMSA shows the binding activities of NF-B in the cells untreated (Cont), treated with 0.1% DMSO alone, and treated with 0.1% DMSO, PSI, MG132, and Lactacystin followed by TNF-␣.
FIG. 6. Proteasome inhibitor Lactacystin prevents TNF-␣– dependent nuclear translocation of p65. Immunocytochemical analysis was performed using an anti-p65 antibody and a FITC-conjugated second antibody. The cells were viewed with a confocal laser scanning microscope at ⫻400 magnification. Cont, the control cells without treatment; TNF, the cells treated with 50 U/ml TNF-␣ for 1 h; TNF/Lacta, the cells pretreated with 10 M Lactacystin for 1 h followed by TNF-␣ for 1 h.
human chondrocyte-like HCS-2/8 cells. It also was shown that IB-␣ in the cytoplasm was degraded rapidly within 15 minutes, and the degradation continued until 3 h after TNF-␣ followed by its restoration at 6 h and 12 h. On the other hand, time course of IB- degradation was different from that of IB-␣. It lasted from 30 minutes to 6 h after TNF-␣. These observations indicate that NF-B activation by TNF-␣ in HCS-2/8 cells consists of three overlapping phases: a rapid activation phase mediated by IB-␣ degradation, a peak activation phase mediated by degradation of both IB-␣ and IB-, and a shutdown phase mediated by restoration of both IBs. Interestingly, duration of NF-B activation by TNF-␣ has been shown to differ among cell types. In Jurkat cells, TNF-␣ induced a rapid and very transient (the peak activation at 30 minutes) activation of NF-B,(16) whereas in human endothelial cells, TNF-␣ caused a rapid but sus-
tained (more than 20 h) activation of NF-B.(35) The difference in time course of NF-B activation was attributed to the variable degradation and synthesis rates of both IB-␣ and IB- among the cell lines. In the Jurkat cells, only IB-␣ was rapidly and transiently degraded, while IB- was not degraded. In contrast, in human endothelial cells, IB-␣ was degraded rapidly and transiently, but the degradation of IB- was sustained for more than 20 h. Collectively, these results indicate that time course of IB-␣ degradation on TNF-␣ is similar in various cells. However, time course of IB- degradation and its restoration differs among cell types. Thus, TNF-␣– dependent degradation and synthesis of IB- seems to determine the duration of NF-B activation. NF-B activation by TNF-␣ in HOS-2/8 cells was associated with marked increase in MMP-1, MMP-3, ICAM-1, and COX-2 gene expression. Treatment with three protea-
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FIG. 7. Proteasome inhibitors attenuate the TNF-␣– dependent increase in MMP-1, MMP-3, ICAM-1, and COX-2 mRNA levels. After the same preincubation as described in Fig. 5, the cells were incubated with TNF-␣ (50 U/ml) for an additional 6 h. Total RNA was extracted, and Northern blot analysis was performed using MMP-1, MMP-3, ICAM-1, COX-2, and GAPDH cDNAs as probes. Representative autoradiographs are shown. After the radioactivities of the bands were measured by the BAS 2000 system, the mRNA levels were normalized by the GAPDH mRNA levels and then expressed as percentage of the level of TNF-␣ alone. The data are expressed as mean ⫾ SD (n ⫽ 4). *p ⬍ 0.05 versus the level of the TNF.
FIG. 8. Dose-dependent inhibition of TNF-␣–induced expression of MMP-1 and MMP-3 by proteasome inhibitor MG132. After 1-h preincubation with various concentrations of MG132, the cells were incubated with TNF-␣ (50 U/ml) for an additional 6 h. Total RNA was extracted, and Northern blot analysis was performed using MMP-1, MMP-3, and GAPDH cDNAs as probes.
some inhibitors, PSI, MG132, and Lactacystin, abrogated the NF-B activation. Compatible with the previous observations,(20,36,37) these inhibitors prevented the degradation of IB-␣. Interestingly, the inhibitors prevented the degradation of phosphorylated as well as nonphosphorylated forms of IB-␣, indicating that they do not interfere the phosphorylation of IB-␣. In addition, it was shown that all of the three inhibitors prevented the degradation of IB-. TNF-␣– dependent induction of MMP-1, MMP-3, ICAM-1,
FIG. 9. Proteasome inhibitors do not affect EGF-dependent expression of MMP-1. After 1-h preincubation with PSI (60 M), MG132 (50 M), or Lactacystin (10 M), the cells were incubated with EGF (10 ng/ml) for 6 h. Northern blot analysis was performed using MMP-1and GAPDH cDNAs as probes.
and COX-2 gene expression also was abrogated by the inhibitors. These results indicate that degradation of IBs followed by the activation of NF-B mediates the induction of these gene expressions in HCS-2/8 cells. Our present study also showed the specificity of proteasome inhibitors to IB degradation, because AP-1– dependent activation of the MMP-1 gene by EGF was not affected by the inhibitors. Involvement of MMP-1, MMP-3, ICAM-1, and COX-2 in the induction of chondrocytic chondrolysis in patients with inflammatory and degenerative joint diseases has been suggested in recent studies. MMP-1 cleaves type II collagen, and MMP-3 decomposes proteoglycan.(38) It was shown that contents of the two MMPs increased in synovial
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fluids of both RA and OA joints,(39 – 41) and thus, they are considered to be key enzymes for the matrix degradation of the cartilage.(42,43) ICAM-1 is a cell surface glycoprotein and acts as a ligand for intercellular communication with lymphocytes.(44) Involvement of ICAM-1 expression in pathogenesis of RA was suggested by a report that treatment with an anti-ICAM-1 antibody was associated with clinical improvement in a group of patients with RA.(45) Also, involvement of COX-2, an inducible prostaglandin G/H synthetase, in pathogenesis of OA was suggested by the observation that its expression and spontaneous release of prostaglandin E2 in ex vivo culture of cartilage specimens from patients with OA were markedly higher than those in normal cartilage.(46) Taken together, the present finding that TNF-␣ via the activation of p65/p50 NF-B induces the expression of MMP-1, MMP-3, ICAM-1, and COX-2 in human chondrocyte-like cells suggests that the activation of NF-B may play an important role in the TNF-␣–induced chondrocytic chondrolysis.
ACKNOWLEDGMENTS This study was funded as a part of the “Ground Research for Space Utilization,” promoted by Japan Space Forum and was supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture, Japan, and by Research Grants for Longevity Sciences from the Ministry of Health and Welfare of Japan.
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Address reprint requests to: Fukushi Kambe, M.D. Department of Endocrinology and Metabolism Research Institute of Environmental Medicine Nagoya University Furo-cho, Chikusa-ku Nagoya 464-8601, Japan
Received in original form May 23, 2000; in revised form December 18, 2000; accepted February 19, 2001.
