QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.103.059220v1 308/2/767    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ahmed, S. Articles by Haqqi, T. M. Search for Related Content PubMed PubMed Citation Articles by Ahmed, S. Articles by Haqqi, T. M. Pubmed/NCBI databases Compound via MeSH Substance via MeSH

INFLAMMATION AND IMMUNOPHARMACOLOGY

Green Tea Polyphenol Epigallocatechin-3-gallate (EGCG) Differentially Inhibits Interleukin-1-Induced Expression of Matrix Metalloproteinase-1 and -13 in Human Chondrocytes

Departments of Medicine (S.A., N.W., M.L., T.M.H.) and Orthopaedics (V.M.G.), Case Western Reserve University School of Medicine, Cleveland, Ohio

Received August 27, 2003; accepted October 31, 2003.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Interleukin-1 (IL-1)-induced inflammatory response in arthritic joints include the enhanced expression and activity of matrix metalloproteinases (MMPs), and their matrix degrading activity contribute to the irreversible loss of cartilage and may also be associated with sustained chronic inflammation. We have earlier shown that green tea (Camellia sinensis) polyphenol epigallocatechin-3-gallate (EGCG) was non-toxic to human chondrocytes [Singh R, Ahmed S, Islam N, Goldberg VM, and Haqqi TM (2002) Arthritis Rheum 46: 2079–2086] and inhibits the expression of inflammatory mediators in arthritic joints [Haqqi TM, Anthony DD, Gupta S, Ahmed N, Lee MS, Kumar GK, and Mukhtar H (1999) Proc Natl Acad Sci USA 96: 4524–4529]. Here we show that EGCG at micromolar concentrations was highly effective in inhibiting the IL-1-induced glycosaminoglycan (GAG) release from human cartilage explants in vitro. EGCG also inhibited the IL-1-induced mRNA and protein expression of MMP-1 and MMP-13 in human chondrocytes. Importantly, EGCG showed a differential, dose-dependent inhibitory effect on the expression and activity of MMP-13 and MMP-1. A similar differential dose-dependent inhibition of transcription factors NF-B and AP-1 by EGCG was also noted. These results for the first time demonstrate a differential dose-dependent effect of EGCG on the expression and activity of MMPs and on the activities of transcription factors NF-B and AP-1 and provide insights into the molecular basis of the reported anti-inflammatory effects of EGCG. These results also suggest that EGCG or compounds derived from it may be therapeutically effective inhibitors of IL-1-induced production of matrix-degrading enzymes in arthritis.

MMPs are a large group of enzymes that play a crucial role in tissue remodeling as well as in the destruction of cartilage and bone in an arthritic joint due to their ability to degrade a wide variety of extracellular matrix components (Mengshol et al., 2002). Production and release of MMPs is microenvironmental and induced by several factors including the proinflammatory cytokine IL-1 (Kraan and van den Berg, 2000). Among the various MMPs, MMP-13 is of particular importance because it is found elevated in joint disorders (Mitchell et al., 1996) and can cleave type II collagen, the major component of the cartilage matrix, more efficiently. Studies have documented that in arthritic joints, degradation of type II collagen is excessive due to increased cleavage by MMPs (Billinghurst et al., 2000). Other studies have shown that excessive activity of MMP-13 can produce the type of pathology seen in arthritic joints (Neuhold et al., 2001). Proinflammatory cytokine-induced expression of MMP-13 in human chondrocytes and in animal models of arthritis is dependent on the activation of the MAPK subgroup JNK and the transcription factor AP-1 (Han et al., 2001; Liacini et al., 2002). In chondrocytes, activation of JNK pathway and Cbfa1 are required for the activation of MMP-13 promoter activity (Mengshol et al., 2001), and indeed, physical interaction between transcription factors Cbfa1 (Runx-2, C/EBP) and AP-1 was shown to be necessary for the activation of MMP-13 promoter (D'Alonzo et al., 2002). An important role of JNK in the pathogenesis of arthritis is also evident from studies showing that inhibitors of JNK protect from arthritis in animal models (Han et al., 2001).

Green tea is a rich source of catechins, and several epidemiological and animal model studies have shown that green tea consumption was associated with health benefits including inhibition of inflammation (Higdon and Frei, 2003). Most of the beneficial health effects of green tea are mimicked by its most prevalent catechin epigallocatechin-3-gallate (EGCG) at micromolar concentrations. EGCG influence a number of cellular mechanisms and has been shown to inhibit the activities of MMP-2 and MMP-9 (Garbisa et al., 2001; Cheng et al., 2003). In addition, EGCG is also an inhibitor of the metallo-elastase and serine-elastase activity and down-regulates the levels of several markers of oxidative stress (Benelli et al., 2002; Dona et al., 2003). We have previously shown that EGCG inhibit the activation of the cytokine-activated JNK and AP-1 pathways in human chondrocytes (Singh et al., 2002). In the present study, we evaluated the potential of EGCG to protect human cartilage explants from IL-1-induced release of cartilage matrix proteoglycans and the induction and expression of MMP-1 and MMP-13 in human chondrocytes.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Reagents. All the culture medium and reagents for molecular biology were obtained from either Cellgro (Mediatech Inc., Herndon, VA) or Invitrogen (Carlsbad, CA). EGCG was purchased from Alexis Biochemicals (San Diego, CA), and recombinant human IL-1 was purchased from R & D Systems (St. Paul, MN). 1,9-Dimethylmethylene blue (DMMB) and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Polyclonal goat anti-human MMP-1 and polyclonal goat anti-human MMP-13 antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Horseradish peroxidase-conjugated anti-goat IgG was purchased from Pierce Biotechnology (Rockford, IL).

Culture of Human OA Cartilage and Chondrocytes. Human OA cartilage samples were procured through the Cooperative Human Tissue Network, with prior approval of the Institutional Review Board of University Hospitals of Cleveland. Full-thickness cartilage slices (20–25 mg) were dissected from the cartilage using sterile scalpel blade (Feather Safety Razor Co., Osaka, Japan). Four to five cartilage pieces (approximately equal in size and weight) were transferred to each well of a 24-well, flat-bottomed plate (NUNC A/S, Roskilde, Denmark) containing DMEM supplemented with antibiotics and 10% fetal calf serum. The cartilage explants were treated with IL-1 alone or with IL-1 + EGCG or EGCG for 72 h. Explants cultured in the absence of IL-1 and EGCG were used as controls. Total glycosaminoglycan present in the culture supernatant was estimated as described below.

Quantitation of Glycosaminoglycans. At the end of the culture period, the culture medium was collected from each group [controls, IL-1 only (10 ng/ml), IL-1 + EGCG (100 µM), and IL-1 + EGCG (200 µM)]. A 50-µl aliquot of the collected supernatant from each sample was used to estimate the total glycosaminoglycan concentration by a colorimetric method employing DMMB as previously described (Farndale et al., 1986). Color intensity was read spectrophotometrically at 535 nm, and the values were derived from a standard curve that was prepared using different concentrations of chondroitin sulfate. Results are expressed as micrograms of glycosaminoglycan released per milligram of cartilage tissue.

Chondrocyte Culture. Chondrocytes were prepared by the enzymatic digestion of femoral head cartilage as previously described (Ahmed et al., 2003). Chondrocytes were plated (1 x 10⁶/ml) in 35-mm culture dishes (Becton-Dickinson, Franklin Lakes, NJ) in complete DMEM with 10% fetal calf serum and allowed to grow for 72 h at 37°C and 5% CO₂ in a tissue culture incubator. They were serum-starved overnight and then treated with IL-1 (5 ng/ml) and IL-1 + EGCG (20–100 µM) for time periods indicated in each figure. Chondrocytes cultured without IL-1 or EGCG served as controls.

Western Immunoblotting for MMP-1 and MMP-13. Confluent chondrocyte cultures were washed with Hank's buffered salt solution and treated with IL-1 in the serum-free medium for 24 h. At the end of the experiments, medium was removed and 500 µl was concentrated using Microcon concentrators (Millipore, Bedford, MA) for 30 min at 25°C. Concentrated samples with equal amounts of protein (25 µg) were mixed with 2x reducing sample buffer and resolved by SDS/PAGE, transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA), and the blot was probed with polyclonal goat anti-human MMP-1 and MMP-13 antibodies (Santa Cruz Biotechnology Inc.). Immunoreactive proteins were visualized by enhanced chemiluminescence using HRP-conjugated anti-goat IgG (Pierce). Images were captured and the intensities of the protein bands were analyzed using the Alpha Innotech Imaging System and are expressed as arbitrary optical density units.

Determination of TIMP-1, MMP-1, and MMP-13 Activity by ELISA. The activities of TIMP-1, MMP-1, and MMP-13 were determined in culture supernatant from the above experiments using commercially available ELISA kits essentially according to the instructions of the manufacturer (Amersham-Pharmacia, Piscataway, NJ) and expressed as Absorbance₄₀₅/h² x 1000.

Quantitative RT-PCR. Total cytoplasmic RNA was prepared from human chondrocytes using a commercially available kit according to the instructions of the manufacturer (Qiagen, Valencia, CA). We used real-time quantitative RT-PCR with internal fluorescent hybridization probes using an ABI Prism 7700 detection system (ABI/Perkin Elmer Biosystems, Foster City, CA) as previously described (Singh et al., 2002). This allows the sensitive and specific quantification of targeted mRNA transcripts. The target-specific RT primer sets and their fluorescent probes used have been described earlier (Singh et al., 2002). The probes were labeled with 5-carbofluorescein (FAM) at the 5' end and with TAMRA at the 3' end (ABI/Perkin Elmer Biosystems). The degradation of the probe during PCR results in increased fluorescence of the probe and allows the detection of the PCR product by monitoring the increase in fluorescence of the reporter dye. To quantitate the expression of MMP-1 and MMP-13 mRNA, single-stranded complementary DNA (cDNA) was synthesized using 100 ng of total RNA prepared from OA chondrocytes as described earlier (Singh et al., 2002). Concentrations of primers and probes were optimized in pilot studies to allow accurate quantitation of the target transcript. The PCR conditions were 1 cycle at 50°C for 2 min, 1 cycle at 95°C, followed by 40 cycles (95°C for 15 s and 60°C for 1 min). To ensure the lack of DNA contamination in the RNA samples, a tube of sample without RT was included as a no-template control. Fourfold serial dilutions of sample cDNAs were used to generate curves of log input versus threshold cycle. Expression of MMP-1 and MMP-13 mRNA was normalized for levels of -actin mRNA and the results are expressed as mRNA copies of MMP/10⁶ copies of -actin mRNA.

Transient Transfection Studies. To study the effect of EGCG on IL-1-induced activation of NF-B and AP-1, human chondrocytes were transiently transfected with reporter plasmids available commercially (Mercury Pathway Profiling System; Clontech, Palo Alto, CA). Briefly, 60 to 80% confluent 1 x 10⁶ cells/35-mm plates were transfected with 1 µg of pNF-B-SEAP and pAP-1-SEAP reporter plasmid or the negative control vector pTAL-SEAP according to the instructions of the manufacturer (Clontech). After transfection, chondrocytes were not treated (controls), treated with IL-1 alone (5 ng/ml), or treated with IL-1 + different concentrations of EGCG. The secreted alkaline phosphatase (SEAP) in the culture medium was detected using the Great Escape SEAP chemiluminescence detection kit (Clontech). The results were expressed as relative light units (RLU) after subtraction of the values obtained with chondrocytes transfected with the negative control vector.

Statistical Analysis. Each experiment was performed three times using cartilage samples from age- and sex-matched donors. Data obtained were pooled, and the level of significance between untreated chondrocytes, chondrocytes treated with IL-1 alone, chondrocytes treated with IL-1 + EGCG, or chondrocytes treated with EGCG alone is based on Dunnett's t test followed by analysis of variance (ANOVA). Values of p < 0.05 were considered significant.

	Results

Top Abstract Materials and Methods Results Discussion References

 
EGCG Inhibited the IL-1-Induced Cartilage Matrix Degradation in Vitro. The effect of EGCG on IL-1-induced cartilage degradation is shown in Fig. 1. Treatment with IL-1 induced the cartilage degradation, measured as the release of glycosaminoglycan in culture medium from cartilage explants, in the culture medium as previously shown (Adcocks et al., 2002). However, the IL-1-induced release of GAG was inhibited in a dose-dependent manner by the green tea catechin EGCG (Fig. 1). The difference in the amounts of glycosaminoglycan released from cartilage explants treated with IL-1 alone and in the two groups treated with IL-1 in the presence of 100 and 200 µM EGCG was statistically highly significant (n = 3, p < 0.005). Untreated cartilage explants also showed a basal level of glycosaminoglycan release but this was also blocked by EGCG indicating that EGCG on its own did not contribute to the enhanced glycosaminoglycan release detected in the group treated with IL-1 alone (Fig. 1). Thus, the critical observation of the present studies is that EGCG appeared to be an effective agent for blocking the IL-1-induced release of glycosaminoglycan from human cartilage explants, at least in vitro.

View larger version (27K): [in this window] [in a new window]   Fig. 1. EGCG inhibited the IL-1-induced release of GAG from human cartilage explants in vitro. Approximately equal size and weight cartilage pieces were incubated in medium alone or medium containing either IL-1 (10 ng/ml) alone, or IL-1 + EGCG (100 µM) or IL-1 + EGCG (200 µM) for 72 h. Total GAG released from cartilage explants in culture supernatant was quantified using a colorimetric method and the values were derived from a standard curve. This experiment was repeated with age- and sex-matched cartilage samples to ensure for the reproducibility. X, p < 0.005 when compared with controls; **, p < 0.005 compared with IL-1 alone treated group; #, p > 0.05 compared with controls; *, p < 0.05 compared with controls and , p < 0.005 compared with IL-1 alone treated group.

EGCG Inhibited the IL-1-Induced Expression of MMP-1, MMP-13, and TIMP-1 in Human Chondrocytes. To evaluate the effect of EGCG on IL-1-induced expression of MMPs protein in human chondrocytes, we treated human chondrocytes with IL-1 alone and with IL-1 + 100 µM EGCG for 24 hrs. As shown in Fig. 2, stimulation of human chondrocytes with IL-1 induced the expression and release of MMP-1 and MMP-13 in the culture supernatant as determined by Western immunoblotting. Analysis of the immunoblot image revealed that the levels of the MMPs detected in the supernatant of chondrocytes treated with IL-1 alone were 4.6-fold higher for MMP-1 and 6.2-fold higher for MMP-13, when compared with the levels detected in untreated chondrocytes (compare lane 1 and lane 2 in each blot). Importantly, cytokine-stimulated increase in the expression of MMP-1 and MMP-13 in the presence of EGCG was inhibited to less than the basal level detected in untreated chondrocyte cultures (p < 0.005). Since MMPs are bound to TIMP-1 as a proenzyme, the production of MMPs may also contribute to the enhanced expression of TIMP-1 and inhibition of MMPs may down-regulate the expression of TIMP-1. To address this possibility, we analyzed the effect of EGCG on IL-1-induced production of TIMP-1 in culture supernatant. As shown in Fig. 3, IL-1 enhanced the production of TIMP-1 in culture supernatant by 36% when compared with the levels detected in untreated chondrocytes (p < 0.05). Prophylactic treatment of chondrocytes with EGCG (100 µM) significantly (p < 0.001) inhibited the IL-1-induced production of TIMP-1 in culture supernatant. Similarly, treatment with EGCG alone also reduced the endogenously released TIMP-1 in chondrocyte cultures as well (p < 0.05). Thus, these data correlate with the down-regulation of IL-1-induced expression of MMPs in human chondrocytes (Fig. 2) and indicate that the effect of EGCG on TIMP-1 expression may not be direct but most likely reflects the inhibitory effect of EGCG on the expression of MMPs.

View larger version (42K): [in this window] [in a new window]   Fig. 2. EGCG inhibited the IL-1-induced expression of MMP-1 and MMP-13 in human chondrocytes. Human chondrocytes were not treated (lane 1), treated with IL-1 alone (lane 2), treated with IL-1 + EGCG (100 µM) (lane 3), or treated with EGCG alone (100 µM) (lane 4) for 24 h. Culture supernatant was concentrated and total proteins (25 µg) were resolved by SDS-PAGE and transferred to nitrocellulose. The Western blot was probed with polyclonal antibodies specific for the human MMP-1 and MMP-13 protein. Results shown are representative of two independent experiments.

View larger version (26K): [in this window] [in a new window]   Fig. 3. EGCG inhibited the IL-1-induced production of TIMP-1 in human chondrocytes. Human chondrocytes (80% confluent) were not treated (1); treated with IL-1 alone (2); treated with IL-1 + EGCG (3–4); or treated with EGCG alone (5) for 24 hr and the TIMP-1 released in the culture medium was measured using an ELISA kit (Biotrak; Amersham). Human chondrocytes stimulated with IL-1 in the presence of EGCG had decreased levels of TIMP-1 (*, p < 0.05; **, p < 0.001) compared with cultures treated with IL-1 alone. Treatment with EGCG alone decreased the constitutive levels of TIMP-1 (#, p < 0.05) when compared with the control values. Values shown are mean ± S.D. of three independent experiments, each performed in triplicate using age- and sex-matched samples.

EGCG Down-Regulated the Activities of MMP-1 and MMP-13 in Human Chondrocytes. We also determined the effect of EGCG on the activities of MMP-1 and MMP-13 using an ELISA kit. To rule out the possibility of detecting cysteine and serine proteinases activity, their inhibitors were added to the conditioned culture medium to abolish their activity prior to the assay. Both MMP-1 and MMP-13 were constitutively active in untreated, control human chondrocytes with the activity level of MMP-1 being at least 3-fold higher than that of MMP-13 (Fig. 4). However, treatment of chondrocytes with IL-1 up-regulated the activities of both the MMP-1 and MMP-13 by 110 and 522% (p < 0.001) when compared with their respective control values (Fig. 4). In this experiment, we also analyzed the dose-dependent effect of EGCG on the activity of MMP-1 and MMP-13. Importantly, treatment of human chondrocytes with IL-1 in the presence of different doses of EGCG (20, 50, or 100 µM) also inhibited the activities of MMP-1 and -13. Compared with human chondrocytes treated with IL-1 alone, the range of percent inhibition by different doses of EGCG was 20 to 97% for MMP-1 and 75 to 93% for MMP-13. Interestingly, this differential inhibitory effect was most pronounced on the activity of MMP-13 when chondrocytes were treated with 20 µM EGCG (Fig. 4) indicating that low doses of EGCG have a marked selective inhibitory effect on the activity of MMP-13 compared with MMP-1, at least in vitro. It is interesting to note that epicatechin or sodium gallate alone or in combination had no inhibitory effect on the activity of either MMP-1 or MMP-13 in this assay (not shown). Additionally, IC₅₀ values for inhibition by EGCG deduced from regression analysis of the dose-response curve for MMP-1 was 27 µM, and for MMP-13 it was 16.5 µM, which is within the physiologically achievable range.

View larger version (24K): [in this window] [in a new window]   Fig. 4. EGCG inhibited the IL-1-induced up-regulation of MMP-13 (A) and MMP-1 (B) activity in human chondrocytes. Human chondrocytes were not treated, treated with 5 ng/ml of IL-1 alone, or treated with IL-1 (5 ng/ml) in the presence of different concentrations of EGCG or with EGCG alone for 24 h and then the supernatant was collected and used to assay for the MMP activity was using MMP-1 and MMP-13 Biotrak ELISA kits (Amersham). Results showed that co-treatment with EGCG significantly inhibited the IL-1-induced up-regulation of both the MMP-13 and MMP-1 activity in a dose-dependent manner (*, p < 0.05, **, p < 0.001). Treatment with EGCG alone showed a marked reduction in the constitutive activity of both the MMPs when compared with the activity levels detected in untreated chondrocytes (#, p < 0.005). Results shown are mean ± S.D. of three independent experiments performed with age- and sex-matched samples.

Inhibition of IL-1-Induced Up-Regulation of MMP-1 and MMP-13 mRNA Expression by EGCG in Human Chondrocytes. The results of the effects of EGCG on the transcription of MMP genes in human chondrocytes are shown in Fig. 5. The level of MMP-1 and MMP-13 mRNAs was quantified by a highly sensitive and specific quantitative RT-PCR method, and the values obtained were normalized to the level of -actin mRNA in the samples. Our results showed that human chondrocytes treated with IL-1 had different levels of the MMP-1 and MMP-13 mRNA (Fig. 5). In all of the samples analyzed, levels of MMP-1 mRNA were higher compared with the level of MMP-13 mRNA in IL-1-stimulated human chondrocytes (Fig. 5). However, mRNA levels showed a marked decline in the samples treated with IL-1 in the presence of EGCG, and this decline was dose-dependent. Interestingly, and in line with the above results, EGCG was most effective against the induction of MMP-13 even at the lowest dose studied whereas higher doses were needed to have an impact on the expression of MMP-1 mRNA. This is a novel observation and has not been previously reported.

View larger version (15K): [in this window] [in a new window]   Fig. 5. EGCG inhibited the IL-1-induced expression of MMP-1 and MMP-13 mRNA in human chondrocytes. Chondrocytes (2 x 106) were stimulated with IL-1 (5 ng/ml) alone or in combination with different concentrations of EGCG for 24 h. Total RNA was prepared, and the expression of MMP-1 and MMP-13 mRNA was determined by real-time quantitative RT-PCR. Values were expressed relative to the levels of -actin mRNA.

Transcription Factors NF-B and AP-1/c-Jun Are Differentially Sensitive to EGCG. In chondrocytes, the gene expression of MMP-13 is tightly regulated by transcription factors NF-B and c-Jun, and inhibition of any one or both could result in the attenuation of MMP-13 (Mengshol et al., 2000). In earlier studies, we have shown that EGCG inhibited the IL-1-induced increase in the nuclear levels of transcription factors NF-B and AP-1/c-Jun in human chondrocytes by blocking their nuclear translocation and activation (Singh et al., 2002a, 2002b; Ahmed et al., 2003). Induction and expression of MMP-13 has been shown to be regulated by the activation of both the transcription factors NF-B and AP-1 (Mengshol, 2000). Using transient transfection with reporter plasmids, we studied whether the inhibition of MMP-1 and MMP-13 mRNA observed in the above experiments could be due to the inhibition of activation and promoter binding activity of NF-B and AP-1 by EGCG. As shown in Fig. 6 (A and B), IL-1 preferentially and strongly stimulated the NF-B pathway (approximately 20 times higher) than AP-1 pathway in transiently transfected chondrocytes. Importantly, our results also showed that EGCG differentially inhibits the promoter activation activity of NF-B and AP-1 and identifies that the activity of transcription factor NF-B was more sensitive to the effect of EGCG, even to the lower doses. Treatment with EGCG inhibited the activity of NF-B by 56 to 94% in a dose-dependent manner (Fig. 7A). In contrast, EGCG at a lower dose (20 µM) had no inhibitory effect on promoter activation by AP-1, and higher concentrations of EGCG (50 and 100 µM) were needed to obtain a 49 to 72% inhibition of AP-1 activity (Fig. 7B). These results correlate with the inhibition of the expression of MMP-1 and MMP-13 mRNAs to the inhibition of NF-B and AP-1. However, the differential dose-dependent effect on the activity of the two transcription factors analyzed in this study suggests that the AP-1-dependent gene expression may not be substantially affected by the physiological concentrations of EGCG. These are also novel findings and have not been reported previously.

View larger version (25K): [in this window] [in a new window]   Fig. 6. Differential sensitivity of the DNA binding activity of transcription factors NF-B and AP-1 to the effects of EGCG in human chondrocytes. Human chondrocytes (60–80% confluent) were transiently transfected with 1 µg each of pNF-B-SEAP (A) and pAP-1-SEAP (B) reported plasmids (Clontech) using the LipofectAMINE reagent. Transfected chondrocytes were then treated with IL-1 alone and in the presence of 20, 50, or 100 µM of EGCG for 24 h. The SEAP was measured in culture supernatant using Chemiluminescence detection kit. (*, p < 0.05; **, p < 0.001 v/s IL-1), and the values are expressed as RLU. Results shown are mean ± S.D. of two independent experiments performed with age- and sex-matched samples. (IL = IL-1; E = EGCG).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Previous work from our laboratory has shown that green tea inhibits the development of arthritis in a mouse model (Haqqi et al., 1999), inhibits the degradation of human cartilage proteoglycan and type II collagen (Adcocks et al., 2002), and selectively inhibits the ADAMTS-1, -4, and -5 (Vankemmelbeke et al., 2003). This study addresses the induction of MMP-1 and MMP-13 in human chondrocytes by IL-1 and demonstrates the ability of EGCG, the most abundant and biologically active catechin of green tea, to inhibit the IL-1-induced cartilage proteoglycan degradation and expression of MMP-1 and MMP-13 in human chondrocytes. Our results not only confirm the previous results (Adcocks et al., 2002) that EGCG blocked the IL-1-induced cartilage proteoglycan release in vitro but extends these further by showing that micromolar concentrations of EGCG effectively inhibit the IL-1-induced up-regulation of MMP-1 and MMP-13 in the human chondrocytes. Almost complete inhibition of both the MMP-1 and MMP-13 enzyme activity was observed at a concentration of 100 µM EGCG. Although this concentration of EGCG may not be achieved physiologically through oral consumption but may readily be achieved through local administration. Our results showing that the two matrix metalloproteinases, MMP-1 and MMP-13, known to be associated with cartilage degradation in an arthritic joint were differentially inhibited by EGCG is an important and novel finding. These results show that MMP-13 was more sensitive to the inhibitory effect of even lower concentrations of EGCG as determined by an ELISA method (Fig. 4). Additionally, and as previously reported (Vankemmelbeke et al., 2003), the inhibitory effect apparently was not via direct inhibition of the activity of MMP-1 or MMP-13 but most likely reflected the inhibition of IL-1-induced expression of their mRNAs suggesting that the effect was at the transcriptional level. This aspect was not addressed in the previous studies (Vankemmelbeke et al., 2003). These findings assign a novel property to EGCG, adding to the anti-cancer and anti-inflammatory properties previously described for this compound (Lin, 2002; Higdon and Frei, 2003). Thus, in addition to blocking the release of proteoglycans from cartilage matrix by inhibiting the ADAMTS (Vankemmelbeke et al., 2003), EGCG also inhibits the IL-1-induced up-regulation of expression and activity of collagenase in physiologically achievable doses in human chondrocytes. Therefore, consumption of green tea or EGCG may inhibit the activities of MMPs involved in the degradation of native collagen, and this may have a suppressive effect on cartilage degradation in arthritic joints.

MMPs are a family of proteolytic enzymes that are normally required for the timely and controlled breakdown of the ECM under normal physiological conditions (Brinckerhoff and Matrisian, 2002). However, their uncontrolled regulation and enhanced expression has been closely associated with the progression of arthritis (Murphy et al., 2002). Earlier studies have shown that EGCG inhibited the gelatinase subgroup of MMPs (MMP-2 and MMP-9) in various cancerous cell lines when stimulated with ultraviolet B radiations, reactive oxygen species (ROS) and pro-inflammatory cytokines like IL-1 and TNF-, thereby inhibiting tumor metastasis (Garbisa et al., 2001; Cheng et al., 2003). However, this is the first elaborative study to evaluate the effect of EGCG on collagenase subgroup of MMPs. Screening of potential molecules possessing possible therapeutic efficiency toward the inhibition of MMPs has recently gained considerable attention (Skiles et al., 2001). Limitations of synthetic MMP inhibitors due to their monomodal nature, lack of specificity, and greater side effects suggest a need to develop therapeutic strategies focusing on the prophylactic agents or supplements that could modify or reverse the progression of cartilage degradation in the affected joints. Recent studies have proved the ability of botanicals in targeting multiple downstream events, alone or with current modalities of treatment to provide higher efficacy and minimal toxicity for an effective intervention (Qiu and Kao, 2003). The catechins are bioavailable following the consumption of green tea with a half-life of a few hours (Yang et al., 1998). Therefore, it is likely that consumption of green tea may have a prophylactic effect on cartilage homeostasis. In this regard, as shown in the present study, green tea catechin EGCG certainly holds promise as it consistently and reproducibly inhibited the IL-1-induced up-regulation of MMPs in human chondrocytes—the only cell type present in the cartilage.

Although these MMPs collectively act in a synchronized manner to degrade articular cartilage, the collagenase subgroup (especially MMP-1 and -13) has been shown to perform a salutary role in chewing the native type II collagen in ECM (Billinghurst et al., 2000). The proteolytic activities of MMPs are precisely regulated during activation from their precursors and inhibition by endogenous inhibitors of metalloproteinases (TIMPs) (Visse and Nagase, 2003). IL-1-induced factors have been shown to activate the MMPs by the disruption of Cys-Zn²⁺ interaction (Brinckerhoff and Matrisian, 2002). Different MMPs have been implicated in different disease conditions, such as gelatinases in cancer metastasis and collagenases in arthritis (Brinckerhoff and Matrisian, 2002). Although, EGCG has been shown to inhibit tumor growth/invasion by inhibiting the gelatinase activity (Garbisa et al., 2001; Cheng et al., 2003), this is the first study showing the effect of EGCG on collagenase gene expression and activity.

There are a number of studies documenting the beneficial health effects of green tea consumption. Most of these studies place emphasis on the anti-cancer properties of green tea, which have now been attributed, at least in part, to the ability of green tea polyphenols to inhibit the gelatinases (Garbisa et al., 2001; Cheng et al., 2003). To this, based on our results, we can add that EGCG in a dose-dependent manner is a potent inhibitor of IL-1-induced induction of collagenases (MMP-1 and MMP-13) and markedly inhibits collagenase-3 (MMP-13) at a physiologically achievable concentration (20 µM) whereas higher concentrations (beyond the concentrations that can be attained simply by drinking green tea) were needed to inhibit the induction and expression of MMP-1 to a meaningful extent. We therefore conclude that inhibition of arthritis following green tea consumption in an animal model (Haqqi et al., 1999) and inhibition of cartilage degradation by EGCG (the present study; Adcocks et al., 2002; Vankemmelbeke, 2003) may be the result of direct inhibition of some of the matrix-degrading enzymes/factors by EGCG through preserving the Cys-Zn²⁺ interaction or in combination with down-regulation of these enzymes/factors at the mRNA level through inhibition of transcription factors. Therefore, it is tempting to suggest that green tea polyphenol EGCG or compounds derived from it may serve as lead agents in the design of more potent and effective inhibitors of collagenases for use therapeutically to block the pathogenesis of arthritis.

	Footnotes

 
This work was supported in part by National Institutes of Health Grants AR-44902, AR-48782, AR-07505, and AR-37726 and funds from the Department of Orthopaedics, Case Western Reserve University.

DOI: 10.1124/jpet.103.059220.

ABBREVIATIONS: IL, interleukin; NO, nitric oxide; MMP, metalloproteinase; MAPK, mitogen-activated protein kinase; JNK, c-Jun NH₂-terminal kinase; AP-1, activating protein-1; EGCG, epigallocatechin-3-gallate; DMMB, 1,9-dimethylmethylene blue; DMEM, Dulbecco's modified Eagle's medium; ELISA, enzyme-linked immunosorbent assay; RT-PCR, reverse transcription-polymerase chain reaction; TAMRA, 5-carboxytetramethylrhodamine; FAM, 5-carbofluorescein; NF-B, nuclear factor-B; RLU, relative light units; TIMP-1, tissue inhibitor of metalloproteinase 1; GAG, glycosaminoglycan; ANOVA, analysis of variance; ROS, reactive oxygen species; PAGE, polyacrylamide gel electrophoresis; SEAP, secreted alkaline phosphatase; OA, osteoarthritis.

Address correspondence to: Dr. Tariq M. Haqqi, Department of Medicine, Division of Rheumatic Diseases, Case Western Reserve University School of Medicine, 2109 Adelbert Rd., Cleveland, OH 44106-4946. E-mail: txh5{at}pop.cwru.edu

	References

Top Abstract Materials and Methods Results Discussion References

 

Adcocks C, Collin P, and Buttle DJ (2002) Catechins from green tea (Camellia sinensis) inhibit bovine and human cartilage proteoglycan and type-II collagen degradation in vitro. J Nutr 132: 341–346.[Abstract/Free Full Text]

Ahmed S, Rahman A, Hasnain A, Goldberg VM, and Haqqi TM (2003) Phenyl-N-tert-butylnitrone down-regulates interleukin-1-stimulated matrix metalloproteinase-13 gene expression in human chondrocytes: suppression of c-Jun NH2-terminal kinase, p38-mitogen-activated protein kinase and activating protein-1. J Pharmacol Exp Ther 305: 981–988.[Abstract/Free Full Text]

Ahmed S, Rahman A, Hasnain A, Lalonde M, Goldberg VM, and Haqqi TM (2002) Green tea polyphenol epigallocatechin-3-gallate inhibits IL-1-induced activity and expression of cyclooxygenase-2 and nitric oxide synthase-2 in human chondrocytes. Free Radic Biol Med 33: 1097–1105.[CrossRef][Medline]

Amin AR and Abramson SB (1998) The role of nitric oxide in articular cartilage breakdown in osteoarthritis. Curr Opin Rheumatol 10: 263–268.[Medline]

Benelli R, Vene R, Bisacchi D, Garbisa S, and Albini A (2002) Anti-invasive effects of green tea polyphenol epigallocatechin-3-gallate (EGCG), a natural inhibitor of metallo and serine proteases. Biol Chem 383: 101–105.[CrossRef][Medline]

Billinghurst RC, Wu W, Ionescu M, Reiner A, Dahlberg L, Chen J, van Wart H, and Poole AR (2000) Comparison of the degradation of type II collagen and proteoglycan in nasal and articular cartilages induced by interleukin-1 and the selective inhibition of type II collagen cleavage by collagenase. Arthritis Rheum 43: 664–672.[CrossRef][Medline]

Blanco FJ and Lotz M (1995) IL-1-induced nitric oxide inhibits chondrocyte proliferation via PGE2. Exp Cell Res 218: 319–325.[CrossRef][Medline]

Brinckerhoff CE and Matrisian LM (2002) Matrix metalloproteinases: a tail of a frog that became a prince. Nat Rev Mol Cell Biol 3: 207–214.[CrossRef][Medline]

Cheng XW, Kuzuya M, Kanda S, Maeda K, Sasaki T, Wang QL, Tamaya-Mori N, Shibata T, and Iguchi A (2003) Epigallocatechin-3-gallate binding to MMP-2 inhibits gelatinolytic activity without influencing the attachment to extracellular matrix proteins but enhances MMP-2 binding to TIMP-2. Arch Biochem Biophys 415: 126–132.[CrossRef][Medline]

D'Alonzo RC, Selvamurugan N, Karsenty G, and Partridge NC (2002) Physical interaction of the activator protein-1 factors c-Fos and c-Jun with Cbfa1 for collagenase-3 promoter activation. J Biol Chem 277: 816–822.[Abstract/Free Full Text]

Dona M, Dell'Aica I, Calabrese F, Benelli R, Morini M, Albini A, and Garbisa S (2003) Neutrophil restraint by green tea: inhibition of inflammation, associated angiogenesis and pulmonary fibrosis. J Immunol 170: 4335–4341.[Abstract/Free Full Text]

Farndale RW, Buttle DJ, and Barrett AJ (1986) Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochem Biophys Acta 883: 173–177.[Medline]

Garbisa S, Sartor L, Biggin S, Salvato B, Benelli R, and Albini A (2001) Tumor gelatinases and invasion inhibited by the green tea flavanol epigallocatechin-3-gallate. Cancer 91: 822–832.[CrossRef][Medline]

Goldring MB, Birkhead JR, Suen LF, Yamin R, Mizuno S, Glowacki J, Arbiser JL, and Apperley JF (1994) Interleukin-1beta-modulated gene expression in immortalized human chondrocytes. J Clin Investig 94: 2307–2316.

Gouze JN, Bordji K, Gulberti S, Terlain B, Netter P, Magdalou J, Fournel-Gigleux S, and Ouzzine M (2001) Interleukin-1beta down-regulates the expression of glucuronosyl-transferase 1, a key enzyme priming glycosaminoglycan biosynthesis: influence of glucosamine on interleukin-1beta-mediated effects in rat chondrocytes. Arthritis Rheum 44: 351–360.[CrossRef][Medline]

Han Z, Boyle DL, Chang L, Bennett B, Karin M, Yang L, Manning AM, and Firestein GS (2001) c-Jun N-terminal kinase is required for metalloproteinase expression and joint destruction in inflammatory arthritis. J Clin Investig 108: 73–81.[CrossRef][Medline]

Haqqi TM, Anthony DD, Gupta S, Ahmed N, Lee MS, Kumar GK, and Mukhtar H (1999) Prevention of collagen-induced arthritis in mice by a polyphenolic fraction from green tea. Proc Natl Acad Sci USA 96: 4524–4529.[Abstract/Free Full Text]

Higdon JV and Frei B (2003) Tea catechins and polyphenols: health effects, metabolism, and antioxidant functions. Crit Rev Food Sci Nutr 43: 89–143.[CrossRef][Medline]

Ismaiel S, Atkins RM, Pearse MF, Dieppe PA, and Elson CJ (1992) Susceptibility of normal and arthritic human articular cartilage to degradative stimuli. Br J Rheumatol 6: 369–373.

Kraan PM and van den Berg WB (2000) Anabolic and destructive mediators in osteoarthritis. Curr Opin Clin Nutr Metab Care 3: 205–211.[CrossRef][Medline]

Liacini A, Sylvester J, Li WQ, and Zafarullah M (2002) Inhibition of interleukin-1-stimulated MAP kinases, activating protein-1 (AP-1) and nuclear factor kappa B (NFkB) transcription factors down-regulates matrix metalloproteinase gene expression in articular chondrocytes. Matrix Biol 21: 251–262.[CrossRef][Medline]

Lin JK (2002) Cancer chemoprevention by tea polyphenols through modulating signal transduction pathways. Arch Pharm Res 25: 561–571.[Medline]

Martel-Pelletier J, McCollum R, DiBattista J, Faure MP, Chin JA, Fournier S, Sarfati M, and Pelletier JP (1992) The interleukin-1 receptor in normal and osteoarthritic human articular chondrocytes. Identification as the type 1 receptor and analysis of binding kinetics and biologic function. Arthritis Rheum 35: 530–540.[Medline]

Mengshol JA, Mix KS, and Brinckerhoff CE (2002) Matrix metalloproteinases as therapeutic targets in arthritic diseases. Arthritis Rheum 46: 13–20.[CrossRef][Medline]

Mengshol JA, Vincenti MP, and Brinckerhoff CE (2001) IL-1 induces collagenase-3 (MMP-13) promoter activity in stably transfected chondrocytic cells: requirement for Runx-2 and activation by p38 MAPK and JNK pathways. Nucleic Acid Res 29: 4361–4372.[Abstract/Free Full Text]

Mengshol JA, Vincenti MP, Coon CI, Barchowsky A, and Brinkerhoff CE (2000) Interleukin-1 induction of collagenase 3 (matrix metalloproteinase 13) gene expression in chondrocytes requires p38, c-Jun N-terminal kinase, and nuclear factor kB. Arthritis Rheum 43: 801–811.[CrossRef][Medline]

Mitchell PG, Magna HA, Reeves LM, Lopresti-Morrow LL, Yocum SA, Rosner PJ, Geoghegan KF, and Hambor JE (1996) Cloning, expression, and type II collagenolytic activity of matrix metalloproteinase-13 from human osteoarthritic cartilage. J Clin Investig 97: 761–768.[Medline]

Murphy G, Knauper V, Atkinson S, Butler G, English W, Hutton M, Stracke J, and Clark I (2002) Matrix metalloproteinases in arthritic disease. Arthritis Res 4: S39–S49.

Neidhart M, Gay RE, and Gay S (2000) Anti-interleukin-1 and anti-CD44 interventions producing significant inhibition of cartilage destruction in an in vitro model of cartilage invasion by rheumatoid arthritis synovial fibroblasts. Arthritis Rheum 43: 1719–1728.[CrossRef][Medline]

Neuhold LA, Killar L, Zhao W, Sung ML, Warner A, Kulik L, Turner J, Wu W, Billinghurst C, Meijers T, et al. (2001) Postnatal expression in hyaline cartilage of constitutively active human collagenase-3 (MMP-13) induces osteoarthritis in mice. J Clin Investig 107: 35–44.[CrossRef][Medline]

Oligino T, Ghivizzani S, Wolfe D, Lechman E, Krisky D, Mi Z, Evans C, Robbins P, and Glorioso J (1999) Intra-articular delivery of a herpes simplex virus IL-1Ra gene vector reduces inflammation in a rabbit model of arthritis. Gene Ther 6: 1713–1720.[CrossRef][Medline]

Pettipher ER, Higgs GA, and Henderson B (1986) Interleukin 1 induces leukocyte infiltration and cartilage proteoglycan degradation in the synovial joint. Proc Natl Acad Sci USA 83: 8749–8753.[Abstract/Free Full Text]

Qiu D and Kao PN (2003) Immunosuppressive and anti-inflammatory mechanisms of triptolide, the principal active diterpenoid from the Chinese medicinal herb Tripterygium wilfordii Hook. f. Drugs R D 4: 1–18.[CrossRef][Medline]

Singh R, Ahmed S, Islam N, Goldberg VM, and Haqqi TM (2002a) Epigallocatechin-3-gallate inhibits interleukin-1-induced expression of nitric oxide synthase and production of nitric oxide in human chondrocytes: suppression of nuclear factor-B (NF-B/p65) activation by inhibiting IB- degradation. Arthritis Rheum 46: 2079–2086.[CrossRef][Medline]

Singh R, Ahmed S, Malemud CJ, Goldberg VM, and Haqqi TM (2002b) Epigallocatechin-3-gallate selectively inhibits interleukin-1-induced activation of mitogen activated protein kinase subgroup c-Jun-N-terminal kinase in human osteoarthritis chondrocytes. J Orthop Res 21: 102–109.

Skiles JW, Gonnella NC, and Jeng AY (2001) The design, structure, and therapeutic application of matrix metalloproteinase inhibitors. Curr Med Chem 8: 425–474.[Medline]

Van de Loo AA and van den Berg WB (1990) Effects of murine recombinant interleukin-1 on synovial joints in mice: measurement of patellar cartilage metabolism and joint inflammation. Ann Rheum Dis 49: 238–245.[Abstract/Free Full Text]

van den Berg WB (2000) Pathophysiology of osteoarthritis. Joint Bone Spine 67: 555–556.[CrossRef][Medline]

Vankemmelbeke MN, Jones GC, Fowles C, Ilic MZ, Handley CJ, Day AJ, Knight CG, Mort JS, and Buttle DJ (2003) Selective inhibition of ADAMTS-1, -4 and -5 by catechin gallate esters. Eur J Biochem 270: 2394–2403.[Medline]

Visse R and Nagase H (2003) Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function and biochemistry. Circ Res 92: 827–839.[Abstract/Free Full Text]

Yang CS, Chen L, Lee MJ, Balentine D, Kuo MC, and Schantz SP (1998) Blood and urine levels of tea catechins after ingestion of different amounts of green tea by human volunteers. Cancer Epidemiol Biomark Prev 7: 351–354.[Abstract]

This article has been cited by other articles:

				S. Ahmed, H. Marotte, K. Kwan, J. H. Ruth, P. L. Campbell, B. J. Rabquer, A. Pakozdi, and A. E. Koch Epigallocatechin-3-gallate inhibits IL-6 synthesis and suppresses transsignaling by enhancing soluble gp130 production PNAS, September 23, 2008; 105(38): 14692 - 14697. [Abstract] [Full Text] [PDF]

				S. Wolfram Effects of Green Tea and EGCG on Cardiovascular and Metabolic Health J. Am. Coll. Nutr., August 1, 2007; 26(4): 373S - 388S. [Abstract] [Full Text] [PDF]

				S. Hsu, D. P. Dickinson, H. Qin, C. Lapp, D. Lapp, J. Borke, D. S. Walsh, W. B. Bollag, H. Stoppler, T. Yamamoto, et al. Inhibition of Autoantigen Expression by (-)-Epigallocatechin-3-gallate (the Major Constituent of Green Tea) in Normal Human Cells J. Pharmacol. Exp. Ther., November 1, 2005; 315(2): 805 - 811. [Abstract] [Full Text] [PDF]

				S. Ahmed, J. Anuntiyo, C. J. Malemud, and T. M. Haqqi Biological Basis for the Use of Botanicals in Osteoarthritis and Rheumatoid Arthritis: A Review Evid. Based Complement. Altern. Med., September 1, 2005; 2(3): 301 - 308. [Abstract] [Full Text] [PDF]

				S. Ahmed, N. Wang, B. B. Hafeez, V. K. Cheruvu, and T. M. Haqqi Punica granatum L. Extract Inhibits IL-1{beta}-Induced Expression of Matrix Metalloproteinases by Inhibiting the Activation of MAP Kinases and NF-{kappa}B in Human Chondrocytes In Vitro J. Nutr., September 1, 2005; 135(9): 2096 - 2102. [Abstract] [Full Text] [PDF]

				X. W. Cheng, M. Kuzuya, K. Nakamura, Z. Liu, Q. Di, J. Hasegawa, M. Iwata, T. Murohara, M. Yokota, and A. Iguchi Mechanisms of the Inhibitory Effect of Epigallocatechin-3-Gallate on Cultured Human Vascular Smooth Muscle Cell Invasion Arterioscler. Thromb. Vasc. Biol., September 1, 2005; 25(9): 1864 - 1870. [Abstract] [Full Text] [PDF]

				H. H. Kim, C. M. Shin, C.-H. Park, K. H. Kim, K. H. Cho, H. C. Eun, and J. H. Chung Eicosapentaenoic acid inhibits UV-induced MMP-1 expression in human dermal fibroblasts J. Lipid Res., August 1, 2005; 46(8): 1712 - 1720. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.103.059220v1 308/2/767    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ahmed, S. Articles by Haqqi, T. M. Search for Related Content PubMed PubMed Citation Articles by Ahmed, S.