Tripterygium wilfordii Hook F Extract Suppresses Proinflammatory Cytokine-Induced Expression of Matrix Metalloproteinase Genes in Articular Chondrocytes by Inhibiting Activating Protein-1 and Nuclear Factor-kB Activities

The major pathologic manifestations of rheumatoid arthritis (RA) and osteoarthritis (OA) are joint inflammation and articular cartilage resorption by proinflammatory cytokine-stimulated matrix metalloproteinases (MMPs) and aggrecanases. The Chinese herbal remedy Tripterygium wilfordii Hook F (TWHF) is effective for treatment of various types of arthritis. However, mechanisms and targets of its actions are poorly understood. Anti-inflammatory activities of the extracts of this plant were previously attributed to inhibition of cyclooxygenase-2 mRNA and prostaglandin E2 synthesis. Here, we show that in primary human femoral head osteoarthritic and normal bovine chondrocytes, TWHF partially or completely inhibited mRNA and protein expression of tumor necrosis factor-a, interleukin (IL)-1, and IL-17-inducible MMP-3 and MMP-13. This agent also inhibited cytokine-stimulated MMP-3 protein expression in human synovial fibroblasts. A dose range of 2.5 to 10 ng/ml of TWHF was effectively inhibitory for IL-1. Pretreatment for 30 min or 1 h (but not 2–10 h) after IL-1 treatment with TWHF inhibited MMP-3 RNA induction. The inhibitory doses had no adverse effect on the viability of chondrocytes. Mechanistic studies revealed no impact on the activation of extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase mitogen-activated protein kinases. Instead, TWHF partially inhibited DNA binding capacity of cytokine-stimulated activating protein-1 (AP-1) and nuclear factor-kB (NF-kB) transcription factors. Therefore, besides its anti-inflammatory activity, this agent may also be effective in blocking cartilage matrix resorption by MMPs by impairing AP-1 and NF-kB binding activities. Thus, TWHF extract contains novel inhibitors of MMP expression that may be of therapeutic potential in arthritis and other conditions associated with increased MMPs. The collagens and aggrecan of cartilage extracellular matrix (ECM) are synthesized by chondrocytes that provide mechanical strength to joints. Rheumatoid arthritis (RA) is a chronic disease with severe joint inflammation, synovial hyperplasia, and deformation. Osteoarthritis (OA) is a prevalent and less inflammatory joint degenerative disease caused by joint overuse, obesity, aging, gender, and cartilage gene mutations (Poole, 1999). Resorption of cartilage is preceded by an initial excessive synthesis of ECM and failure of repair processes (Aigner and Dudhia, 1997) induced by an imbalance between anabolic growth factors (transforming growth factor-b and insulin-like growth factor-I) and proinflammatory cytokines, interleukin (IL)-1, and tumor necrosis factor-a (TNF-a) (Dinarello and Moldawer, 1999). These cytokines induce matrix metalloproteinases (MMPs) that release cartilage ECM fragments, which serve as markers of arthritis (Lark et al., 1997). MMPs include matrilysin, stromelysins, gelatinases, interstitial and neutrophil collagenases, collagenase-3 (MMP-13), and membrane-type MMPs (Westermarck and Kähäri 1999). They digest different components of the ECM during physiologic and pathologic turnover. Stromelysin-1 (MMP-3) cleaves proteoglycans, collagens, gelatins, and link protein of aggrecan. An imbalance between active MMPs and the tissue This research was supported by the Arthritis Society of Canada and in part by the Canadian Institutes of Health Research and the Canadian Arthritis Network (CAN). ABBREVIATIONS: ECM, extracellular matrix; RA, rheumatoid arthritis; OA, osteoarthritis; IL, interleukin; TNF-a, tumor necrosis factor-a; MMP, matrix metalloproteinase; TWHF, Tripterygium wilfordii Hook F; COX, cyclooxygenase; AP-1, activator protein-1; NF-kB, nuclear factor-kB; DMEM, Dulbecco’s modified Eagle’s medium; FCS, fetal calf serum; PBS, phosphate-buffered saline; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MTT, (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide; EMSA, electrophoretic mobility shift assay; PEA-3, polyomavirus enhancer A binding protein-3; BSA, bovine serum albumin. 0026-895X/01/5905-1196–1205$3.00 MOLECULAR PHARMACOLOGY Vol. 59, No. 5 Copyright © 2001 The American Society for Pharmacology and Experimental Therapeutics 778/898172 Mol Pharmacol 59:1196–1205, 2001 Printed in U.S.A. 1196 at A PE T Jornals on A uust 7, 2017 m oharm .aspeurnals.org D ow nladed from inhibitors of metalloproteinases in OA cartilage contributes to its breakdown (Dean et al., 1989). The MMPs and aggrecanase cleaves aggrecan at distinct sites (Fosang et al., 1996; Tortorella et al., 1999). Increased MMP-3 in the serum and synovium of RA patients is a marker of inflammation (Yoshihara et al., 1995). MMP-3 was localized in the superficial zone of cartilage and in the synovium of patients with OA (Okada et al., 1992). We showed that 50% of the OA patients had elevated MMP-3 mRNA in their synovium (Zafarullah et al., 1993). IL-1 increased MMP-3 expression in rabbit cartilage (Hutchinson et al., 1992). A 20-fold excess of MMP-3 over MMP-1 was reported in the synovial fluids of patients with RA (Walakovits et al., 1992). In human cartilage, MMP-3 mRNA was preferentially increased compared with MMP-1 in the presence and absence of IL-1 (Nguyen et al., 1992). MMP-13 produced by chondrocytes enhances cleavage and denaturation of type II collagen in OA cartilage (Mitchell et al., 1996; Billinghurst et al., 1997) and aggrecan at the MMP-specific site and a new site (Fosang et al., 1996). Its expression is increased in rheumatoid synovium (Lindy et al., 1997), OA cartilage (Reboul et al., 1996), and is further induced by IL-1 and TNF-a via c-fos mediation (Borden et al., 1996). Like other MMPs, the secreted inactive pro-MMP-13 is activated extracellularly by gelatinase A and MT1-MMP (Knäuper et al., 1996). IL-1 and TNF-a are the main proinflammatory cytokines in joints that suppress ECM synthesis and increase MMPsmediated cartilage resorption (Dinarello and Moldawer, 1999). IL-17 is a newer proinflammatory cytokine in synovial fluid (Dinarello and Moldawer, 1999) that induces IL-1b, IL-6, and stromelysin in chondrocytes (Shalom-Barak et al., 1998). Inhibition of IL-1 and TNF-a actions by IL-1 receptor antagonist via gene therapy and antibodies to TNF-a are beneficial in reducing the symptoms of arthritis (Feldmann et al., 1997; Oligino et al., 1999). Thus, blocking IL-1-, TNFa -, and IL-17-induced MMP gene expression by novel physiologic and pharmacologic inhibitors (Vincenti et al., 1994; Lark et al., 1997) is an important therapeutic approach for arthritis. The Chinese herbal remedy Tripterygium wilfordii Hook F (TWHF) (Lei Gong Teng or thunder god vine) has been known for thousands of years as a therapeutic agent against arthritis and autoimmune diseases (Lipsky and Tao, 1997). This agent has both immunosuppressive and anti-inflammatory activities, including inhibition of cytokine gene expression in T cells (Tao et al., 1996). The anti-inflammatory actions have been attributed to the inhibition of cyclooxygenase (COX)-2 and prostaglandin E2 in rheumatoid fibroblasts and other cell types (Tao et al., 1998). We examined whether TWHF extract was also effective against major MMPs involved in the resorption of arthritic cartilage. Here, we show for the first time that TWHF potently inhibits proinflammatory cytokine-induced MMP-3 and MMP-13 gene expression partly by interfering with DNA binding activities of AP-1 and NF-kB transcription factors. Furthermore, MMPs are novel targets of antiarthritic actions of TWHF. Materials and Methods Reagents. Dulbecco’s modified Eagle’s medium (DMEM), fetal calf serum (FCS), penicillin streptomycin, Fungizone, and agarose were from Canadian Life Technologies, Inc. (Burlington, ON, Canada). One hundred-millimeter plates and T-75 flasks were from Nunc (Roskilde, Denmark). IL-1b, TNF-a, and IL-17 were obtained from R&D Systems (Minneapolis, MN). Collagenase type II was from Sigma-Aldrich (Oakville, ON, Canada). The chemiluminescence systems were from Roche Molecular Biochemicals (Laval, PQ, Canada). Zeta-probe and nitrocellulose membranes were from Bio-Rad Canada (Mississauga, ON, Canada). RNA probe labeling kits were from Promega (Madison, WI). Restriction endonucleases, T7 polymerase, and RNase inhibitor were from Amersham Pharmacia Biotech (Baie d’urfé, PQ, Canada). TWHF pills (containing 33 mg of TWHF per pill) were obtained from Huangshi Pharmaceuticals (Hubei Province, Peoples Republic of China), dissolved in 100% ethanol, filter-sterilized, and stored at 2 20°C. Primary Cultures of Bovine and Human Chondrocytes, Synovial Fibroblasts, and Treatments. Normal bovine articular cartilage was obtained from the knee joints of adult animals from a local abattoir. Human cartilage was from the femoral heads of the OA patients who underwent hip replacement surgery at Notre Dame Hospital. Chondrocytes were released by pronase (1 mg/ml) for 60 min and collagenase (Sigma type IA) digestions for 9 h in DMEM at 37°C. Cells were washed five times with phosphate-buffered saline (PBS) and grown in DMEM with 10% FCS as high-density primary monolayer cultures until confluent growth. Cells were distributed in 6-well plates, grown to confluence, washed with PBS, kept in serumfree DMEM for 24 h, TWHF extract added at the final concentrations of 2.5 and 5 ng/ml (or as indicated) 30 min before treatment with IL-1b (10 ng/ml), TNF-a (20 ng/ml), and IL-17 (20 ng/ml) for 24 h. Human knee synovial fibroblasts at passage 13 were maintained in medium with 0.5% FCS and were subjected to the same treatments. RNA Extraction and Northern Hybridization Analysis. Total RNA was extracted by the guanidinium isothiocyanate procedure (Chomczynski and Sacchi, 1987) and aliquots of 3 to 5 mg analyzed by electrophoretic fractionation in 1.2% formaldehyde-agarose gels. The integrity and quantity of RNA were verified by ethidium bromide staining of the 28S and 18S ribosomal RNA bands. RNA was transferred completely onto Zeta-probe nylon membrane with a Bio-Rad Transblot in the presence of 0.5 3 Tris/acetate/EDTA buffer at a current of 500 mA for 12 h. Northern blots were hybridized with a human stromelysin cDNA probe generously provided by Dr. Richard Breathnach (Nantes, France). This probe, which cross-hybridizes with the bovine stromelysin RNA, was a 1.6-kilobase pair EcoRI cDNA fragment cloned in the plasmid pGEM-4Z (Promega Biotech) and the vector linearized with NarI. A 491-base-pair reverse transcriptase-polymerase chain reaction collagenase-3 cDNA product (Shlopov et al., 1997) was cloned in pGEM-4Z, identified by DNA sequencing, and linearized with EcoRI. The human 28S ribosomal RNA plasmid (American Type Culture Collection, Manassas, VA) was digested with XbaI. All antisense RNA probes were synthesized with T7 polymerase according to the protocols of Promega Biotech and labeled to high-specific activity (1 3 10 cpm/mg) with [a-P]CTP (3000 Ci/mmol; DuPont Canada, Inc., Mississauga, ON,