Anti-arthritis effects of vitamin K 2 (menaquinone-4) ) a new potential therapeutic strategy for rheumatoid arthritis Hiroshi Okamoto, Kumi Shidara, Daisuke Hoshi and Naoyuki Kamatani Institute of Rheumatology, Tokyo Women’s Medical University, Japan Vitamin K is a generic term for compounds that include phytonadione (vitamin K 1 ), the menaquinone series (vitamin K 2 ) and menadione (vitamin K 3 ). These vitamin K compounds share a common chemical struc- ture consisting of a naphthoquinone nucleus. Vita- min K 1 has a long phytol side chain whereas vitamin K 2 has an unsaturated side chain [1]. Vitamin K 2 acts as a cofactor for a vitamin K- dependent carboxylase involved in the carboxylation of coagulation factors and is an essential substrate for blood coagulation [2]. It has been reported that osteo- porosis and fractures frequently occurred after the long-term use of warfarin, which inhibits the effect of vitamin K upon coagulation [3]. Vitamin K 2 has been shown to be a key inducer of bone mineralization in human osteoblasts and has also been reported to inhi- bit osteoclastogenesis of bone by induction of the osteoclast apoptosis [4–6]. Human studies have demonstrated that vitamin K 2 is proposed to be an effective treatment for osteoporo- sis and the prevention of fractures [7]. Menaquinone-4 (MK-4), the most common form of vitamin K 2 is frequently used for the treatment of osteoporosis in Japan and other Asian countries. In addition to these biological activities, vitamin K 2 has been reported to exert an inhibitory effect on the growth of several cell lines and tumor cells such as hepa- toma cells [8]. Several lines of evidence indicate that vitamin K 2 has a potent pro-apoptotic effect on leuke- mia cell lines and primary cultured leukemia cells [9,10]. In addition, several case studies have demonstrated the clinical benefit of vitamin K 2 in the treatment of patients with acute myeloid leukemia and myelodysplastic syn- drome [11–13]. Thus far, there are no studies examining the effect of vitamin K 2 on animal models of inflamma- tory arthritis or humans with inflammatory arthritis. Keywords apoptosis; collagen type II-induced arthritis; rheumatoid arthritis; vitamin K 2 (menaquinone-4) Correspondence H. Okamoto, Institute of Rheumatology, Tokyo Women’s Medical University, 10–22 Kawada-cho, Shinjuku, Tokyo 162–0054, Japan Fax: +81 3 5269 1726 Tel: +81 3 5269 1725 E-mail: hokamoto@ior.twmu.ac.jp (Received 9 December 2006, revised 26 June 2007, accepted 12 July 2007) doi:10.1111/j.1742-4658.2007.05987.x Vitamin K 2 (menaquinone-4, MK-4) has been reported to induce apoptosis in hepatocellular carcinoma, leukemia and myelodysplastic syndrome cell lines. The effects of MK-4 on the development of arthritis have never been addressed thus far. In the present study, we investigated the effect of MK-4 upon the proliferation of rheumatoid synovial cells and the development of arthritis in collagen-induced arthritis. We analyzed the effect of MK-4 on the proliferation of fibroblast-like synoviocytes using the 3-(4,5-de- methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The pro-apop- totic effect of MK-4 upon fibroblast-like synoviocytes was investigated with annexin V staining and DNA fragmentation and caspase 3 ⁄ 7 assays. Moreover, we analyzed the effect of MK-4 on the development of colla- gen-induced arthritis in female dark agouti rats. Our results indicated that MK-4 inhibited the proliferation of fibroblast-like synoviocytes and the development of collagen-induced arthritis in a dose-dependent manner. We conclude that MK-4 may represent a new agent for the treatment of rheu- matoid arthritis in the setting of combination therapy with other disease- modifying antirheumatic drugs. Abbreviations CIA, collagen-induced arthritis; CII, collagen type II; FLS, fibroblast-like synoviocytes; MK-4, menaquinone-4; MTT, 3-(4,5-demethylthiazol-2- yl)-2,5-diphenyl-tetrazolium bromide; RA, rheumatoid arthritis; TNF-a, tumor necrosis factor a. 4588 FEBS Journal 274 (2007) 4588–4594 ª 2007 The Authors Journal compilation ª 2007 FEBS In the present study, we determined the effect of vitamin K 2 (MK-4) on the proliferation of rheumatoid synovial cells and the development of arthritis in the experimental model of collagen type II-induced arthri- tis (CIA). Results Effect of MK-4 on the viability of fibroblast-like synoviocytes It has been reported that MK-4 can induce apoptosis in several tumor cells. It has been also reported that rheumatoid arthritis (RA) synovial cells proliferate as fierce as tumor cells [14]. We hypothesized that MK-4 could also reduce the viability of synovial cells and thus be a novel treatment for RA because the marked proliferation of synovial cells is a key pathological fea- ture of RA. We therefore studied the biological effects of MK-4 on the proliferation of fibroblast-like syno- viocytes (FLS). We observed that MK-4 inhibited the viability of FLS in a dose-dependent manner (Fig. 1). By contrast, vitamin K 1 , which exerts no inhibitory effects on the proliferation of tumor cell lines [10], had no significant effect on the viability of synovial cells. These results indicate that MK-4 exerts cytotoxic effects on synovial cells and this may be secondary to its side chain structure. Induction of apoptosis of fibroblast-like synoviocytes by MK-4 Analogous to the effects of MK-4 on tumor cells, we hypothesized that MK-4 induces apoptotic death of synovial cells. We therefore examined the level of apoptosis in synovial cells by measuring annexin V staining, DNA fragmentation and caspase activity. As shown in Fig. 2A, most of the cells treated for 30 min with MK-4 (10 )6 m) exhibited diffuse cytosolic annexin V staining (mean ± SD; 45 ± 9 ⁄ 100 cells) compared with positive (63 ± 11 ⁄ 100 cells) and negative (12 ± 4 ⁄ 100 cells) controls. To further confirm the pro-apoptotic effect of MK-4 on FLS, we conducted a DNA fragmentation assay with various concentrations of MK-4. MK-4 exhibited dose-dependent pro-apopto- tic effects on FLS (Fig. 2B). To determine whether MK-4 activates caspase 3 and 7 to induce apoptosis on FLS, we conducted a caspase activity assay. MK-4 activated caspase assay in a dose-dependent manner (Fig. 2C). By contrast, vitamin K 1 did not show any effects on caspase activity and DNA fragmentation (data not shown). These results indicate that the inhib- itory effect of MK-4 on the proliferation of FLS is secondary to the induction of apoptosis by MK-4 induction of caspase activation. Oral administration of MK-4 ameliorates collagen-induced arthritis As shown in Fig. 3, MK-4 suppressed it suppressed the initiation of clinical arthritis. compared with con- trol rats treated with NaCl ⁄ P i , as demonstrated by paw volume (Fig. 3A), arthritis score (Fig. 3B) and bone destruction score (Fig. 3C). MK-4 treated rats exhibited statistically significant effects in a dose- dependent manner (P<0.01). Histological analysis of the ankle joints of MK-4-treated rats (10 mgÆkg )1 Æ day )1 and 50 mgÆkg )1 Æday )1 groups) at day 32 demon- strates that MK-4 inhibits synovial proliferation and pannus formation compared with control rats (NaCl ⁄ P i ) (Fig. 3D). Histology of a joint of a rat of similar age is also shown in Fig. 3D. There was no 0 25 50 75 100 125 0 25 50 75 100 125 Cell Viability (%) Cell Viability (%) Vitamin K2 (MK-4) 0 * ** * * ** * 52.51.250.630.3130.15 Vitamin K1 0 10 (x10 -7 M) 10 (x10 -7 M)52.51.250.630.3130.15 Fig. 1. Inhibition of synovial cell viability by vitamin K 2 (MK-4). The inhibitory effect of vitamin K 2 and vitamin K 1 was evaluated with the MTT assay. The level of the absorbance in the MTT assay from untreated cells was taken as 100%. The data are presented as the mean ± SD. H. Okamoto et al. Anti-arthritis effects of vitamin K 2 FEBS Journal 274 (2007) 4588–4594 ª 2007 The Authors Journal compilation ª 2007 FEBS 4589 Annexin V-FITC Propidium IodideFITC + PI Annexin V-FITC Propidium IodideFITC + PI Annexin V-FITC Propidium IodideFITC + PI A Positive control (TNF-α) MK-4 Negative control (NaCl/P i ) 0 0.4 0.8 1.2 MK-4 DNA fragmentation (absorbance A 270 nm ) 0 60 Caspase 3/7 activity (luminescence (RLU)) MK-4 B C * ** * ** 0 10 (x10 -7 M)52.51.250.63 TNF-αIL-1β 40 20 * 0 10 (x10 -7 M)52.51.250.630.31 ** * * * TNF- α Fig. 2. Induction of apoptosis in FLS by MK-4. (A) FLS were cultured on 2-well Labo- ratory-Tek tissue culture chamber slides. Cells were double-stained with annexin V and propidium iodine using the ApoAlert Annexin V-fluorescein isothiocyanate apop- tosis kit. In positive controls, cells were treated with 1 ngÆmL )1 of TNF-a. In negative controls, cells were treated with NaCl ⁄ P i . (B) DNA fragmentation induced by MK-4 was determined with the Cellular DNA Frag- mentation ELISA kit. In positive controls, cells were treated with 1 ngÆmL )1 of TNF-a. In negative controls, cells were treated with NaCl ⁄ P i . Each measurement was performed in triplicate and the results are presented as the absorbance (A 270 nm ) compared with positive (TNF-a) and negative (NaCl ⁄ P i ) con- trols. (C) Caspase activity induced by MK-4 was determined using the Caspase-Glo 3 ⁄ 7 Assay kit. In positive controls, cells were treated with 1 ngÆmL )1 of TNF-a or 1ngÆmL )1 of interleukin-1b. In negative con- trol, cells were treated with NaCl ⁄ P i . Each measurement was performed in triplicate and the results are presented as the lumi- nescence (relative light units) compared with positive (interleukin-1b and TNF-a) and negative (NaCl ⁄ P i ) controls. *P<0.05, **P<0.01 versus control (–), by the paired t-test. Anti-arthritis effects of vitamin K 2 H. Okamoto et al. 4590 FEBS Journal 274 (2007) 4588–4594 ª 2007 The Authors Journal compilation ª 2007 FEBS mortality or weight loss in MK-4-treated rats. These data suggest that MK-4 has significant inhibitory effects on arthritis in vivo because MK-4 suppressed the initiation of arthritis in the CIA model. Discussion Synovial hyper-proliferation has reported to be caused, at least in part, by impaired apoptosis of FLS. Defi- cient apoptosis of FLS results from up-regulation of anti-apoptotic molecules such as bcl-2, sumo-1 and FLIP (Fas-associated death domain-like interleukin 1b converting enzyme inhibitory protein) [15–18]. Defi- cient apoptosis of FLS was reported to result from lower expression of pro-apoptotic molecules such as PTEN (phophatase and tensin homologue deleted from chromosome 10) [19,20]. These data suggested that the synovial hyperplasia in RA is the result of defective apoptosis not due to antibody or complement media- tiated cytotoxicity. In support of this model, various compounds, including antirheumatic drugs, could induce apoptosis in FLS. Indeed, methotrexate, hydroxychloroquine, and bucillamine have all been reported to cause apoptosis in FLS [21–24]. Therefore, a compound that induces apoptosis in FLS could potentially be efficacious in the treatment of RA. Vitamin K 2 is significantly less toxic than other anti- proliferative agents such as methotrexate. In addition, vitamin K 2 has significant anti-osteoporotic effects. Thus, vitamin K 2 may well open up novel future strat- egies, including chemoprevention, for the management of patients with RA. Vitamin K 2 is an established treatment for patients with osteoporosis in Japan and has been used for more than 10 years [7]. In the pres- ent study, we have shown that vitamin K 2 inhibited the proliferation of FLS through the induction of apoptosis and also inhibited the development of CIA in a dose-dependent manner. Therefore, vitamin K 2 2 2.5 3 3.5 4 4.5 0 10 20 30 Days 0 0.5 1 1.5 2 2.5 0 10 20 30 Days 0 0.5 1 1.5 2 Tarsal bone Metatarsal bone Calcanus 50mg/kg10mg/kgControlNormal MK-4 Score of Bone Destruction Paw volume (ml) Arthritis score 50mg/kg 10mg/kg Control Normal Control (NaCl/P i ) Normal MK-4 10mg/kg MK-4 50mg/kg AB CD * * * * * * * * * * * Fig. 3. Suppression of arthritis development by MK-4 in the collagen type-II induced arthritis model. (A,B) MK-4 suppressed the progression of clinical arthritis compared with control rats treated with NaCl ⁄ P i , as demonstrated by paw volume and the clinical arthritis score. The data are represented as the mean ± SD. *P < 0.05. (C) Radiological examination of bone destruction at the affected joints (calcaneus, metatarsal and tarsal bone) in normal rats, MK-4-treated rats (10 mgÆkg )1 Æday )1 ), MK-4 treated rats (50 mgÆkg )1 Æday )1 ) and control rats (NaCl ⁄ P i )as described in Experimental procedures. (D) Histological findings of the foot joint in normal rats, MK-4-treated rats (10 mgÆkg )1 Æday )1 ), MK-4-treated rats (50 mgÆkg )1 Æday )1 ) and control rats (NaCl ⁄ P i ). H. Okamoto et al. Anti-arthritis effects of vitamin K 2 FEBS Journal 274 (2007) 4588–4594 ª 2007 The Authors Journal compilation ª 2007 FEBS 4591 may represent a new strategy for the treatment of RA, presumably in the setting of combination therapy with other disease-modifying antirheumatic drugs. Further clinical studies are needed to evaluate the beneficial effects of vitamin K 2 . Experimental procedures Synovial fibroblasts Synovial biopsy samples were obtained from six patients with RA and synoviocytes were maintained in separate cul- tures. These patients had active RA as defined by the clini- cal criteria of the American Rheumatism Association [25]. All RA patients were receiving treatment that included methtrexate (8 mgÆweek )1 ) and nonsteroidal anti-inflamma- tory drugs, as well as steroids (less than 5 mgÆday )1 ). How- ever, patients treated with biological agents, such as tumor necrosis factor a (TNF-a) blocking agents, were excluded from the study. All experiments were carried out using cell cultures dur- ing the third to seventh passage. FLS were cultured at 37 °Cin5%CO 2 in DMEM (Nikken Bio Medical Labora- tory, Kyoto, Japan) supplemented with 10% fetal bovine serum (Bioscience International Inc., Rockville, MD, USA). Reagents Vitamin K 1 (2-methyl-3-phenyl-1,4-naphthoquinone) and vitamin K 2 (MK-4) were all supplied by EisaiCo., Ltd. (Tokyo, Japan). MTT assay FLS were seeded at a density of 1 · 10 3 cells per well in 96- well microtiter plates in 100 lL serum-free DMEM and were treated with various concentrations of MK-4 (1.56 · 10 )7 m to 10 )6 m) for 48 h. Cell proliferation was evaluated by measuring the number of viable cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay [26]. Experiments were performed six times with each of the three independent cell cultures. Annexin V staining To examine annexin V staining, FLS were cultured on 2-well Laboratory-Tek tissue culture chamber slides. After treatment with MK-4 (10 )6 m), cells were fixed with 4% paraformaldehyde in NaCl ⁄ P i for 15 min at room tempera- ture and examined by microscopy. Cells were double- stained with annexin V and propidium iodine using the ApoAlert Annexin V-fluorescein isothiocyanate apoptosis kit (Takara Bio Inc., Shiga, Japan). DNA fragmentation assay Apoptosis was determined using the Cellular DNA Frag- mentation ELISA kit (Roche Diagnostics, Mannheim, Ger- many) to detect BrdU labeled DNA fragments. Each measurement was performed in triplicate and the results are presented as the absorbance (A 270 nm ) compared with posi- tive (TNF-a) and negative (NaCl ⁄ P i ) controls. Caspase 3 ⁄ 7 assay Apoptosis was determined using the Caspase-Glo 3 ⁄ 7 assay kit (Promega Co., WI, USA) to detect caspase 3 ⁄ 7 activity. Each measurement was performed in triplicate and the results are presented as the luminescence (relative light units) compared with positive (interleukin-1b and TNF-a) and negative (NaCl ⁄ P i ) controls. CIA model in rats Seven-week-old female dark agouti rats were obtained from Japan SLC, Inc. (Shizuoka, Japan). 0.3% Collagen type II (CII) solution (Koken-Cellgen, Koken, Co. Tokyo, Japan) was used. CII emulsion was made by mixing 1 mL of 0.9% saline, 3 mL of incomplete Freund’s adju- vant and 2 mL of 0.3% CII solution. CIA was induced by an intradermal injection of 200 lL of CII emulsion (collagen II 1 mgÆmL )1 ) at the base of the tail. Treatment with MK-4 was commenced at the onset of the disease. MK-4 and control NaCl ⁄ P i were orally administered once per day at the specified dose for 32 days. Each group was comprised with ten female dark agouti rats. MK-4 was freshly suspended in 0.5% methyl cellulose diluted in NaCl ⁄ P i . In each experiment, a group of control rats were administered 1% methyl cellulose orally. Rats were examined for signs of CIA at days 1, 7 (onset of arthri- tis), 14, 18, 21, 25, 28 and 32 after immunization using the clinical parameters of paw swelling and clinical score. The footpad volume was measured with a plethysmome- ter TK-101 (Unicom Japan, Tokyo, Japan). A scoring system from 0–4 was used for the clinical evaluation of CIA as follows: 0, normal; 1, mild swelling; 2, moderate swelling; 3, severe swelling; 4, severe and non-weight- bearing arthritis. Each limb was graded giving a maximal clinical score of 4 per animal [27]. For histological evalu- ation, we performed hematoxylin and eosin staining of tissue specimens of the ankle in five rats in both groups. Radiological examination of bone destruction at the affected joints (calcaneus, metatarsal and tarsal bone) of two rats in both group was graded from 0–3 as follows: 0, normal; 1, minor signs of destruction; 2, up to 30% destruction; 3, more than 30% destruction [27]. There was no mortality and no body weight loss in MK-4-trea- ted rats. Anti-arthritis effects of vitamin K 2 H. Okamoto et al. 4592 FEBS Journal 274 (2007) 4588–4594 ª 2007 The Authors Journal compilation ª 2007 FEBS Statistical analysis The Mann–Whitney U-test was used to compare nonpara- metric data for statistical significance. This test was used to evaluate the histological examination of ankle joints, paw volume and the clinical arthritis score. Acknowledgements The expert technical help of Yukiko Katagiri is grate- fully acknowledged. 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Arthritis Rheum 31, 315–324. 26 Okamoto H, Cujec TP, Okamoto M, Peterlin BM, Baba M & Okamoto T (2000) Inhibition of the RNA-depen- dent transactivation and replication of human immuno- deficiency virus type 1 by a fluoroquinoline derivative K-37. Virology 272, 402–408. 27 Larsson E, Harris HE, Palmblad K, Mansson B, Saxne T & Klareskog L (2005) CNI-1493, an inhibitor of pro- inflammatory cytokines, retards cartilage destruction in rats with collagen induced arthritis. Ann Rheum Dis 64, 494–496. Anti-arthritis effects of vitamin K 2 H. Okamoto et al. 4594 FEBS Journal 274 (2007) 4588–4594 ª 2007 The Authors Journal compilation ª 2007 FEBS . Anti -arthritis effects of vitamin K 2 (menaquinone- 4) ) a new potential therapeutic strategy for rheumatoid arthritis Hiroshi Okamoto, Kumi Shidara, Daisuke Hoshi and Naoyuki Kamatani Institute. conducted a caspase activity assay. MK-4 activated caspase assay in a dose-dependent manner (Fig. 2C). By contrast, vitamin K 1 did not show any effects on caspase activity and DNA fragmentation (data. unsaturated side chain [1]. Vitamin K 2 acts as a cofactor for a vitamin K- dependent carboxylase involved in the carboxylation of coagulation factors and is an essential substrate for blood coagulation