CTGF/Hcs24 induces chondrocyte differentiation through a p38 mitogen-activated protein kinase (p38MAPK), and proliferation through a p44/42 MAPK/extracellular-signal regulated kinase (ERK) Gen Yosimichi 1,2 , Tohru Nakanishi 1 , Takashi Nishida 1,3 , Takako Hattori 1 , Teruko Takano-Yamamoto 2 and Masaharu Takigawa 1,3 1 Department of Biochemistry and Molecular Dentistry, and 2 Department of Orthodontics, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan; 3 Biodental Research Center, Okayama University Dental School, Japan Connective tissue growth factor/hypertrophic chondrocyte specific gene product 24 (CTGF/Hcs24) promotes pro- liferation and differentiation of chondrocytes in culture. We investigated the roles of two major types of mitogen acti- vated protein kinase (MAPK) in the promotion of prolif- eration and differentiation by CTGF/Hcs24. Here we report the effects of the MAPKK/MEK 1/2 inhibitor, PD098059, and p38 MAPK inhibitor, SB203580, in a human chondro- sarcoma-derived chondrocytic cell line (HCS-2/8) and rabbit growth cartilage (RGC) cells treated with CTGF/ Hcs24. In the proliferation phase, CTGF/Hcs24 induced a < fivefold increase in the phosphorylation of p44/42 MAPK/ERK and a < twofold increase in that of p38 MAPK in an in vivo kinase assay. These inhibitors of MAPKK and MAPK suppressed phosphorylation of ets-like gene-1 (Elk-1) and nuclear activating transcription factor-2 (Atf-2) induced by CTGF/Hcs24 in a dose-dependent manner, respectively. Western blot analysis showed that phosphorylation of ERK was induced from 30 to 60 min and phosphorylation of p38 MAPK from 10 to 15 min after the addition of CTGF/Hcs24 in confluence HCS-2/8 cells. PD098059 suppressed the DNA synthesis of HCS-2/8 cells and RGC cells, while SB203580 did not. On the other hand, the p38 MAPK inhibitor, SB203580, completely inhibited the CTGF/Hcs24-induced synthesis of proteoglycans in HCS-2/8 cells and RGC cells but the MEK1/2 inhibitor, PD098059, did not. These results suggest that ERK mediates the CTGF/Hcs24-induced proliferation of chondrocytes, and that p38 MAPK mediates the CTGF/Hcs24-induced differentiation of chondrocytes. Keywords: connective tissue growth factor; hypertrophic chondrocyte specific gene product (CTGF/Hcs24); MAPK; chondrocyte; MAPK inhibitor; signal transduction. Connective tissue growth factor/hypertrophic chondrocyte specific gene product 24 (CTGF/Hcs24) is a cysteine-rich, heparin-binding protein. Its gene and two other genes, cef10/ cyr61 and nov, belong to the CCN gene family [1 –5]. Recently, new members of this family, wisp2/rcop-1 [6], wisp3/elm1 [7,8], and ctgf-L [9], were isolated, but the functions of these genes are unknown. We cloned a mRNA preferentially expressed in chondrocytes from a human chondrosarcoma-derived chondrocytic cell line, HCS-2/8 [10,11] by differential display-PCR [12]. The gene product was identical to CTGF. CTGF/Hcs24 is strongly expressed on hypertrophic chondrocytes in growth plate of cartilage [12], and promotes proliferation and differentiation of chon- drocytes in culture [13]. In addition, CTGF/Hcs24 promotes both proliferation and differentiation of osteoblasts [14]. Although a CTGF/Hcs24-specific receptor [15] is yet to be cloned, a CTGF/Hcs24-receptor complex with an apparent molecular mass of 280 kDa was tyrosine-phophorylated in HCS-2/8 cells (data not shown). CTGF/Hcs24 has also multiple effects on fibroblasts [16], endothelial cells [17,18], and tumor cells [19]. CTGF/Hcs24 is highly expressed in the process of wound healing, and mediates fibrotic disorder [4,17,18]. CTGF/Hcs24 and Cyr61 are potential angio- genetic factors [3 –5,18], and directly bind to integrin a V b 3 and a IIb b 3 on fibloblasts [20,21]. CTGF/Hcs24 and Cyr61 induce adhesion of human fibloblasts mediated by integrin a 6 b 1 and cell surface heparan sulfate proteoglycans, and activate intracellular signaling molecules including focal adhesion kinase (FAK), paxillin, and Rac, and sustained phosphorylation of ERK [22]. Recently, the transcriptional mechanism of CTGF/Hcs24 was revealed [23], and it was also reported that intracellular CTGF/Hcs24 may act as an antimitotic agent [24]. But the mechanisms of the multiple functions of CTGF/Hcs24 described above are not well understood. MAPK pathways are essential mitogenic pathways in many cell lines and responsible for various growth factors. MAPK is activated by a wide variety of growth factors such Correspondence to M. Takigawa, Department of Biochemistry and Molecular Dentistry, Graduate school of Medicine and Dentistry, Okayama University Dental School, 2-5-1 Shikata-cho Okayama 700-8525, Japan. Fax: 1 81 86 2356649, Tel.: 1 81 86 235 6645, E-mail: takigawa@md.okayama-u.ac.jp (Received 21 May 2001, revised 1 August 2001, accepted 21 September 2001) Abbreviations: CTGF/Hcs24, connective tissue growth factor/ hypertrohic chondrocyte specific gene product 24; Elk-1, ets-like gene-1; Atf-2, nuclear activating transcription factor-2; DMEM, Dulbecco’s modified Eagle’s medium; aMEM, alpha minimal essential medium; MAPK, mitogen-activated protein kinase; ERK, extracellular- signal regulated kinase; HCS-2/8, human chondrosarcoma-derived chondrocytic cell line clone 2/8; RGC, rabbit growth cartilage cells; GDF-5, growth differentiation factor-5; PTH, parathyroid hormone; EGF, epidermal growth factor. Eur. J. Biochem. 268, 6058–6065 (2001) q FEBS 2001 as epidermal growth factor (EGF) [25], nerve growth factor (NGF) [26], fibroblast growth factor (FGF) [27], and trans- forming growth factor (TGF)-b [28,29], and directs phos- phorylation of transcription factors, such as ets-like gene-1 (Elk-1) [30], nuclear activating transcription factor-2 (Atf-2) [31], and c-Jun [32], and other kinases. MAPK cascades are composed of many kinds of kinases and are intricately regulated. However, downstream of these pathways can simply be classified into three major groups mediated by the following kinases; p44/42 MAPK/ERK, p38 MAPK, and c-jun N-terminal kinase (JNK). It is reported that Elk-1 and Atf-2 are phosphorylated by ERK and p38 MAPK, respec- tively [30,33,34]. In this study, we investigated the signal transducible pathways of CTGF/Hcs24 responsible for its multiple roles in the proliferation and differentiation of chondrocytes, and analyzed the relationship between the MAPK pathways and the proliferation and differentiation of CTGF/Hcs24, using two MAPK inhibitors, the MEK1/2-specific inhibitor, PD098059 [35,36], and the p38 MAPK inhibitor, SB203580 [37,38] MATERIALS AND METHODS Cell culture and materials A human chodrosarcoma-derived chondrocytic cell line, HCS-2/8, was inoculated at a density of 2 Â10 4 per cm 2 in 96-well plates, 24-well plates, and six-well plates (Sumitomo Bakelite Co. Ltd, Tokyo, Japan) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Nissui Pharmaceutical Co. Ltd, Tokyo, Japan) containing 10% fetal bovine serum (Upsdate Biotechnology, Inc., Lake Placid, NY, USA), and 50 ng : mL 21 of human recombinant CTGF/ Hcs24 (rCTGF/Hcs24). For the inhibitor assay, 50 m M of MEK1/2 inhibitor (PD098059, Calbiochem, San Diego, CA, USA), or 10 m M of p38 MAPK inhibitor (SB203580, Calbiochem) was added to the culture after 24 h of serum depletion, and cells were harvested after 12 h of incubation. Rabbit growth cartirage (RGCs) cells were isolated from growth cartilage of ribs of young rabbits as described previously [13,39,40]. The isolated cells were inoculated at a density of 1 Â10 4 per cm 2 in 96-well plates, 24-well plates, and six-well plates (Sumitomo Bakelite Co. Ltd), and cultured in aMEM (alpha minimal essential medium, ICN Biomedicals, Inc., Costa Mesa, CA, USA) containing 10% fetal bovine serum. In vivo MAP kinase luciferase assay ERK phosphorylation was quantified using an MAPK in vivo kinase assay kit and p38K in vivo kinase assay kit (Clontech) according to the manufacturer’s protocol. This system is designed to detect endogeneous ERK and p38 MAPK activity in vivo. A pTet-Elk or pTet-Atf vector expresses a fusion protein with the functional domain of Elk or Atf and Tet repressor (TetR) domain. The reporter vector, pTRE-Luc, contains a tet-responsive element (TRE) upstream of the luciferase gene. Phosphorylation of Elk by ERK, or Atf by p38 MAPK causes homodimeriization of these proteins that induces DNA binding through the TRE element, and results in the activation of the reporter gene. For this experiment, 3 Â10 5 HCS-2/8 cells in a 35-mm tissue culture dish were transiently cotransfected with 1 mg of pTRE-luc, 1 mg of pTet-Elk or pTet-Atf by FuGENE TM 6 (Roche, Indianapolis, IN, USA). An internal control plasmid, 0.5 mg of pRL-TK, was also cotransfected for monitoring transfection efficiency. At 24 h post-transfection, the cells were incubated with various concentrations of PD098059 or SB203580 for 1 h, and cells were cultured with or without 50 ng : mL 21 of rCTGF/Hcs24 for 24 h. The cell lysates were prepared and assayed for luciferase activity using the Dual-Luciferase TM Reporter Assay System (Promega) according to the manufacturer’s instructions. Light emission was measured for 12–24 s with a luminometer (TD-20/20: Tuner Design, Sunnyvale, CA, USA). Western blotting Total cellular protein was prepared by lysing cells in lysis buffer [20 m M Tris/HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 mM Na (VO 4 ), 5% glycerol, 40 m M ammonium molybdate, and 1 mM phenylmethane- ulfonyl flyoride]. Then, 4 mg of the protein was separated by 4–20% gradient SDS/PAGE and electrophoretically trans- ferred to poly(vinylidene difluoride) filters (Bio-Rad). The filters were blocked with 5% nonfat drymilk in Tris-buffered saline (pH 7.5) containing 0.1% Tween 20 for 30 min at room temparature and then incubated with anti-ERK Ig, anti-(active-ERK) Ig, anti-(p38 MAP) Ig or anti-(active-p38 MAPK) Ig (New England BioLaboratories, Bevely, MA, USA, Calbiochemistry, San Diego, CA, USA and Santa Cruz Biochemistry, Santa Cruz, CA, respectively) for 24 h at 4 8C. The filters were then incubated with the secondary antibodies [horseradish peroxidase-conjugated anti-(rabbit IgG) Ig (DAKO, Trappes, France), and alkaline phospha- tase-conjugated anti-(rabbit IgM) Ig (Cappel, Durham, NC, USA)], and the signal was detected by enhanced chemiluminescence (Amersham Pharmacia), or colored with nitro blue tetrazolium and 5-bromo-4-chloroindolyl phosphate (BCIP). DNA synthesis For measurement of DNA synthesis, HCS-2/8 cells were inoculated at a density of 2 Â 10 4 per well in 96-well multiplates with 100 mL of DMEM containing 10% fetal bovine serum. When they reached subconfluence, the medium was changed to fetal-bovine-serum-free DMEM and preincubated for 24 h. In the case of RGC cells, the cells were inoculated at a density of 1 Â 10 4 per well in 100 mLof aMEM containing 10% fetal bovine serum. When they reached confluence, the medium was changed to fetal- bovine-serum-free aMEM, and preincubated for 48 h. MAPK inhibitors (50 m M of PD098059, 10 mM of SB203580) were added to the culture 1 h before the addition of 50 ng : mol 21 of rCTGF/Hcs24. After 22 h, [ 3 H]thymidine (9925 Tbq : nmol 21 ; Amersham Pharmacia Biotech), dissolved in DMEM was added to the cultures at a final concentration of 740 KBq : mL 21 , and cells were incubated for another 4 h. After labeling, the cell layers were washed three times with NaCl/P i and treated successively with 5% trichloroacetic acid and ethanolethyl ether (3 : 1, v/v). Radioactivity in the residual materials was measured using a Micro b-PLUS (Pharmacia Biotech), as described previously [13,15]. q FEBS 2001 MAPK mediates CTGF actions on chondrocytes (Eur. J. Biochem. 268) 6059 Proteoglycan synthesis Proteoglycan synthesis was assayed as described previously [10,15] with slight modification. HCS-2/8 cells were grown to confluence in 24-well microplates in DMEM containing 10% fetal bovine serum. They were then preincubated in DMEM without fetal bovine serum for 24 h and incubated in the same medium with rCTGF/Hcs24 (50 ng : mL 21 ) for 5 h. Then, [ 35 S]sulfate (37 MBq : mL 21 ) dissolved in NaCl/ P i was added to the cultures to a final concentration of 370 kBq : mL 21 , and incubation was continued for another 17 h. After labeling, the cultures were digested with 1mg : mL 21 actinase E (Kaken Pharmaceuticals, Tokyo, Japan), and the radioactivity of the material precipitated with cetylpyridium chloride was measured in a scintillation counter. Statistical analysis Unless otherwise specified, all experiments were repeated at least twice, with similar results. One-way analysis of variance ( ANOVA) was used for statistical evaluation. Statistical analysis was performed by the Dunnett test if necessary. Data are expressed as the mean ^ SD and P , 0.05 was considered significant. RESULTS In vivo MAP kinase and p38 MAPK luciferase assay HCS-2/8 cells were transiently cotransfected with an Elk or Atf expression vector, pTet-Elk or pTet-Atf, together with a pTRE-luc plasmid that contains a luciferase reporter gene. Transfected cells were subsequently treated with various concentrations of rCTGF/Hcs24. The luciferase activity in this experiment reflects endogeneous ERK and p38 MAPK phosphorylation activity. The luciferase activity increased on incubation with rCTGF/Hcs24 in a dose-dependent manner (Fig. 1). A high dose of CTGF/Hcs24 (50 ng : mL 21 ) caused a fivefold increase in the MAPK luciferase activity (Fig. 1A), but the same concentration of rCTGF/Hcs24 caused only a twofold increase in the p38 MAPK luciferase activity (Fig. 1B). To check the phosphorylation of Elk by ERK and Atf by p38 MAPK through the activation of MEK1/2 and p38 MAPK, we used PD098059, a MEK1/ 2-specific inhibitor and SB203580, a p38 MAPK inhibitor in the same assay (Fig. 2). Phosphorylation of Elk (Fig. 2A) or Atf (Fig. 2B) was suppressed by each inhibitor dose- dependently. Effects of rCTGF/Hcs24 on phosphorylation of ERK and p38 MAP kinase in HCS-2/8 cells We analyzed the effects of rCTGF/Hcs24 on the phosphory- lation of two major types of MAP kinases, ERK and p38 MAP kinase, by Western blotting in HCS-2/8 cells (Fig. 3). We determined that CTGF/Hcs24 stimulated the phosphory- lation of the kinases with different time kinetics. Interest- ingly, CTGF/Hcs24 induced a slow phosphorylation of ERK from 10 min after the treatment with a maximal effect observed at 30 min (Fig. 3A). This was different from other growth factors that promote proliferation. On the other hand, rCTGF/Hcs24 induced a rapid phosphorylation of p38 MAPK from 10 to 30 min after the addition (Fig. 3B). The MEK1/2-specific inhibitor, PD098059, and p38 MAPK- specific inhibitor, SB203580, inhibited the phosphorylation of each MAP kinase induced by rCTGF/Hcs24 (Fig. 4A,B). But neither inhibitor had an effect on the different phosphorylation of MAP kinase. Effects of MAP kinase inhibitors on DNA synthesis in HCS-2/8 and RGC cells To investigate the signal pathways for proliferation of chondrocytes induced by CTGF/Hcs24, we performed DNA Fig. 1. In vivo MAPK and p38 MAPK luciferase activity induced by CTGF/Hcs24 in the sparse phase of HCS-2/8 cells. Elk-1 (A) and Atf-2 (B) phosphorylation induced by CTGF/Hcs24 through endo- geneous ERK and p38 MAPK, respectively. HCS-2/8 cells were cotransfected with the fusion transactivater plasmid, pTet-Elk (A) and pTet-Atf (B) and the reporter plasmid, pTRE-luc, as described in Materials and methods. At 24 h after transfection, the cells were serum- deprived for 24 h, and incubated with the indicated concentrations of CTGF/Hcs24 for 24 h. Extracts prepared thereafter were assayed for luciferase activity using the Dual-Luciferase TM Reporter Assay System. In these system, firefly luciferase is used as an expression reporter of TRE (pTRE-Luc), and TK drived renilla luciferase is usd as a transfection control (pRL-TK). These plasmids were cotransfected to HCS-2/8 cells. F/R ratio indicates the ratio of luciferase activity of firefly (F) and renilla (R). Points and bars are the mean ^ SD for duplicate cultures. *, P , 0.05; **, P , 0.01 (significantly different from the control culture). 6060 G. Yosimichi et al. (Eur. J. Biochem. 268) q FEBS 2001 synthesis analysis by estimating incorporation of [ 3 H]thymi- dine in HCS-2/8 (Fig. 5A) and RGC cells (Fig. 5B). rCTGF/ Hcs24 at a concentration of 50 ng : mL 21 induced about a 1.5-fold increase in DNA synthesis both in HCS-2/8 and in RGC cells (Fig. 5A,B, bar 2) compared to the control culture (Fig. 5A,B, bar 1). In HCS-2/8 cells, treatment with 50 m M of the MEK1/2 inhibitor, PD098059, suppressed the degree of DNA synthesis in the control culture by about half, either with (Fig. 5A, bar 3) or without rCTGF/Hcs24 (Fig. 5A, bar 5). On the other hand, 10 m M of the p38 MAPK inhibitor, SB203580 suppressed DNA synthesis in HCS-2/8 cells induced by CTGF/Hcs24, but had no effect in the control culture (Fig. 5A, bars 1 and 6) without rCTGF/ Hcs24 (Fig. 5A, bar 6). In RGC cells (Fig. 5B), rCTGF/ Hcs24 induced about a 1.5-fold increase in the synthesis of DNA similar (Fig. 5B, bar 2) to the control (Fig. 5B, bar 1). PD098059 inhibited the effect of CTGF/Hcs24 (Fig. 5B, bar 3), but had no effect on the basal level of DNA synthesis in RGC cells (Fig. 5B, bar 5). SB203580 treatment did not suppress the synthesis of DNA in RGC cells with or without rCTGF/Hcs24 (Fig. 5B, bar 6). SB203580 did not decrease the synthesis further in PD098059-treated cells (Fig. 5B, bar7). Effects of MAP kinase inhibitors on proteoglycan synthesis of HCS-2/8 and RGC cells Previously we reported that CTGF/Hcs24 dose-dependently enhanced proteoglycan synthesis in HCS-2/8 cells and RGC cells [13]. To investigate the intracellular pathways for the stimulatory action of CTGF/Hcs24 on chondrocyte differ- entiation, we examined proteoglycan synthesis in HCS-2/8 cells (Fig. 6A) and RGC cells (Fig. 6B) in the presence of MAP kinase inhibitors. Addition of 50 ng : mL 21 of rCTGF/ Hcs24 promoted the synthesis of proteoglycans by about twofold in both HCS-2/8 cells (Fig. 6A, bar 2) and RGC cells (Fig. 6B, bar 2) relative to control cultures (Fig. 6A,B, bar 1). Treatment with 50 m M of the MEK1/2 inhibitor, PD098059 did not inhibit the stimulatory effect of rCTGF/ Hcs24 (Fig. 6A,B, bar 3), but 10 m M of the p38 MAPK inhibitor, SB203580 suppressed the stimulatory effects of rCTGF/Hcs24 in HCS-2/8 cells (A bar 4) and RGC cells (B bar 4). The inhibitors had no effect on the basal level of Fig. 3. Effects of CTGF/Hcs24 on phosphorylation of ERK (A) and p38 MAPK (B) in confluent HCS-2/8 cells. Western blot analysis revealed that stimulation with CTGF/Hcs24 increased the phosphoryl- ation of ERK from 10 min to 30 min (A). Stimulation with CTGF/ Hcs24 also increased the phosphorylation of p38 MAPK peaked from 10 to 30 min (B). Fig. 2. Effects of MAP kinase inhibitors (PD098059, SB203580) on phosphorylation of Elk-1 and Atf-2 induced by CTGF/Hcs24. HCS- 2/8 cells were cultured under the same conditions as for Fig. 1. CTGF/ Hcs24 (50 ng : mL 21 ) was added after 1 h pretreatment with various concentrations of MAP kinase inhibitors (PD098059, A; SB203580, B). CTGF/Hcs24-induced phosphorylation of Elk-1 (A) and Atf-2 (B) through endogeneous ERK was suppressed by PD098059 (A) and SB203580 (B), respectively, dose-dependently. F/R ratio indicates the ratio of luciferase activity of firefly (F) and renilla (R). Points and bars are the mean ^ SD for duplicate cultures. *, P , 0.05; **, P , 0.01 (significantly different from the control culture). Fig. 4. Effects of MAPK inhibitors (50 mM PD098059 and 10 mM SB203580) on phosphorylation of ERK (A) and p38 MAPK (B) determined by Western blotting. (A) Phosphorylation of ERK was enhanced by CTGF/Hcs24 at 30 min. Treatment with 50 m M PD098059 decreased the amount of phosphorylated ERK but treatment with 10 m M SB203580 had no effect. (B) Phosphorylated p38 MAPK was increased at 15 min and 30 min, and the increase was suppressed by SB203580. PD098059 treatment had no effect on the amount of phosphorylated p38 MAPK. q FEBS 2001 MAPK mediates CTGF actions on chondrocytes (Eur. J. Biochem. 268) 6061 proteoglycan synthesis in HCS-2/8 cells (Fig. 6A, bars 5 and 6) and RGC cells (Fig. 6B, bars 5 and 6). DISCUSSION In this study, we first investigated the phosphorylation of two major types of MAP kinase induced by CTGF/Hcs24. The MAPK cascade is known to be activated by receptor tyrosine kinase activity induced by growth factors, such as EGF [35], FGF [27], and parathyroid hormone (PTH) [41] and serine threonine kinase activity by TGF-b [28,29] and growth differentiation factor-5 (GDF-5) [42] in chondro- cytes. We previously reported that a 280-kDa CTGF/ Hcs24-receptor complex was present in HCS-2/8 cells [14], and a human osteosarcoma cell line, Saos-2 [15], and this receptor complex was thyrosine-phosphorylated by CTGF/ Hcs24 (data not shown). It was also reported that Nov, a member of the CCN family, induced thyrosine-phosphoryl- ation of a 221-kDa protein in fibloblasts [43]. These Fig. 5. Effects of MAP kinase inhibitors on DNA synthesis in HCS-2/8 (A) and RGC cells (B). For the evaluation of DNA synthesis, HCS-2/8 cells were inoculated at a density of 2 Â 10 4 per well into 96-well multiplates and cultured in 100 mL of DMEM containing 10% fetal bovine serum, while RGC cells were inoculated at a density of 1 Â 10 4 per well into 96-well multiplates and cultured in 100 mLof aMEM containing 10% fetal bovine serum. When they reached subconfluence, HCS-2/8 cells were incubated in 100 mL of serum-free DMEM for 24 h, and RGC cells were incubated in 100 mL of serum- free aMEM for 24 h, and then 50 ng : mL 21 of CTGF/Hcs24 was added to the cultures. Next, 50 m M PD098059 and 10 mM SB203580 were added 1 h before the addition of rCTGF/Hcs24. Dimethylsulfoxide was added to the control culture. DNA synthesis was measured 22 h later as described in Materials and methods. (A) control (bar 1), 50 ng : mL 21 of CTGF/Hcs24 (bar 2), 50 m M PD098059 with 50 ng : mL 21 of rCTGF/ Hcs24 (bar 3), 10 m M SB203580 with 50 ng : mL 21 of CTGF/Hcs24 (bar 4), 50 m M PD098059 (bar 5), 10 mM SB203580 (bar 6), both of the inhibitors with 50 ng : mL 21 of rCTGF/Hcs24 (bar 7). Columns and bars are mean ^ SD for triplicate cultures. **, P , 0.01, significantly different from the control cultures. *, P , 0.05, significantly different from the CTGF/Hcs24 containing cultures. Fig. 6. Effects of MAP kinase inhibitors on proteoglycan synthesis in HCS-2/8 (A) and RGC cells (B). For estimation of proteoglycan synthesis, confluent cultures of HCS-2/8 cells (A) and RGC cells (B) were preincubated in serum-free DMEM for 24 h and then incubated in the same medium with or without 50 ng : mL 21 of rCTGF/ Hcs24. Next, 50 m M PD098059 and 10 mM SB203580 was added 1 h before the addition of rCTGF/Hcs24. Dimethylsulfoxide was added to the control culture. and then [ 35 S]sulfate (37 Mbq : mL 21 ) dissolved in NaCl/P i was added to the cultures (370 kBq : mL 21 final concentration), and incubation was continued for another 17 h. Proteoglycan synthesis was measured as described in Materials and methods. Control (bar 1), 50 ng : mL 21 of rCTGF/Hcs24 (bar 2), 50 m M PD098059 with 50 ng : mL 21 of rCTGF/Hcs24 (bar 3), 10 mM SB203580 with 50 ng : mL 21 of rCTGF/Hcs24 (bar 4), 50 mM PD098059 (bar 5), 10 mM SB203580 (bar 6), both of the inhibitors with 50 ng : mL 21 of rCTGF/Hcs24 (bar 7). Columns and bars are mean ^ SD for duplicate cultures. **, P , 0.01, significantly different from the control cultures. *, P , 0.05, significantly different from the CTGF/Hcs24-added cultures. 6062 G. Yosimichi et al. (Eur. J. Biochem. 268) q FEBS 2001 observations indicate a possible relation between CTGF/ Hcs24-signal transduction and MAPK cascades. The present study demonstrated that CTGF/Hcs24 potentiated phosphory- lation of Elk-1, downsream of ERK [30], and Atf-2, downstream of p38 MAPK [31] in a dose-dependent manner in HCS-2/8 cells (Fig. 1). Both ERK1 and ERK2 have been shown to be activated by their upstream activaters, MEK1 and MEK2. A recently developed MEK1/2 inhibitor, PD098059, has been reported to bind to MEK1/2 and inhibit MEK1/2 phosphorylation activated by either c-Raf or MEKK [35,36]. PD098059 efficiently and specifically suppresses the activation of ERK in response to various growth factors [25,26,28,44]. The inhibitory effect of PD098059 on MEK2 is less potent than that on MEK1 (IC 50 values of PD098059 for MEK1 and MEK2 are 4 and 50 m M, respectively) [35]. On the other hand, p38 MAPK is known to be a stress signal transducer, and phosphorylation of p38 MAPK in chondro- cytes by EGF [25], PTH [41], and GDF-5 [42] regulates differentiation of chondrocytes. p38 MAPK is phosphory- lated by MKK3, and MKK6 [45], and Atf-2 is one of the transcription factors downstream of p38 MAPK [31]. A specific p38 MAPK inhibitor, SB203580 [38], suppresses two isoforms of p38 MAPK, p38a,andb2, but not p38g [37,46]. In this study, the MEK1/2 inhibitor, PD098059, dose- dependently suppressed phosphorylation of ERK, and Elk-1 downstream of ERK in the growth phase of HCS-2/8 cells treated with CTGF/Hcs24 (Fig. 2). Similarly, the p38 MAPK inhibitor, SB203580, suppressed phosphorylation of p38 MAPK, and Atf-2 downstream of p38 MAPK (Fig. 2). Western blot analysis revealed that CTGF/Hcs24 increased phosphorylation of ERK and p38 MAPK in confluent HCS- 2/8 cells (Fig. 3). PD098059 suppressed the phosphoryl- ation of ERK, but had no effect on the phosphorylation of p38 MAPK. In the same way, SB203580 suppressed phosphorylation of p38 MAPK but not ERK (Fig. 4). These results indicate that both MAPK pathways contributed selectively to the effects of CTGF/Hcs24. Next, we showed the effects of MAP kinase inhibitors in both HCS-2/8 cells and RGC cells induced by CTGF/Hcs24. Previously, we reported that CTGF/Hcs24 induced DNA synthesis, and proteoglycan synthesis in HCS-2/8 cells and RGC cells [13]. We investigated the effects of MAP kinase inhibitors on pathways of proliferation by analyzing DNA synthesis, and pathways of differentiation by analyzing proteoglycan synthesis, in HCS-2/8 and RGC cells. In the case of HCS-2/8 cells, the MEK1/2 inhibitor, PD098059, suppressed the synthesis of DNA under all conditions, and the p38 MAPK inhibitor, SB203580, suppressed the syn- thesis induced by CTGF/Hcs24 to the control level (Fig. 5A, bar 4), but had no effect on the basal level (Fig. 5A, bar 6). As HCS-2/8 cells were tumor cells, their basal levels of DNA synthesis were high, and proliferation was not under normal control. Therefore we analyzed normal chondrocytic response in RGC cells, PD098059 suppressed DNA syn- thesis induced by CTGF/Hcs24 to the control level (Fig. 5B, bar 3) and had no effect on the basal level. SB203580 did not suppress DNA synthesis induced by CTGF/Hcs24, and had no effect in the absence of CTGF/Hcs24 (Fig. 5B, bars 3 and 5). These findings suggest that the ERK pathway has an important role in chondrocyte proliferation induced by CTGF/Hcs24. Next, we analyzed proteoglycan synthesis to investigate the effects of MAP kinase inhibitors on the differentiation of chondrocytes induced by CTGF/Hcs24. We previously reported that CTGF/Hcs24 induced synthesis of proteo- glycans in HCS-2/8 cells and RGC cells [13]. In the present study, as shown in Fig. 6, PD098059 had no effect on proteoglycan synthesis in the presence or absence of CTGF/ Hcs24 (Fig. 6A,B, bars 3 and 5). On the other hand, SB203580 inhibited the increase in proteoglycan synthesis evoked by CTGF/Hcs24 in HCS-2/8 and RGC cells (Fig. 6A,B bar 4). These findings suggest that the p38 MAPK pathway is the most important signalling pathway in the differentiation of chondrocytes. The ERK pathway does not seem to be involved in the differentiation of chondrocytes especially when induced by CTGF/Hcs24. Concerning crosstalk between ERK and p38 MAPK, it is reported that GDF-5 phosporylated p38 MAPK and ERK, and p38 MAPK inhibitor inhibited, but MEK1/2 inhibitor enhanced, the chondrogenic response induced by GDF-5 in ATDC5 cells [42]. EGF inhibited the chondrogenic differen- tiation of mesenchymal cells, and activated ERK but inhibited p38 MAPK [25]. Related to the effect of CTGF/ Hcs24 on chondrocytes, luciferase assay revealed phosphory- lation of Elk-1 was inhibited by the MEK inhibitor but slightly enhanced by treatment with the p38 MAPK inhibitor (data not shown). and phosphorylation of Atf-2 was inhibited by the p38 MAPK inhibitor and also inhibited slightly by the MEK inhibitor (data not shown). These results suggest that ERK activates both Elk-1 and Atf-2 (Fig. 7), and has a dominant effect in the growth phase of cells. Furthermore, DNA synthesis indicated that both the ERK and p38 MAPK pathways are involved in the proliferation of chondrocytes induced by CTGF/Hcs24 in tumor cells. Also Western blotting shows that SB203580 treatment increased phos- phorylation of ERK (Fig. 4, at 0 min), but the effect was not related to synthesis of DNA or proteoglycan (Figs 5 and 6, bars 5 and 6). These results suggest that inhibition of one pathway induces activation of another. In summary, the present results emphasized the functional importance of two MAP kinase cascades, the ERK and p38 MAPK pathways, in the promotion of the chondrogenesis Fig. 7. Possible scheme of MAPK signaling cascades in CTGF/ Hcs24-induced chondrogenesis. E, extracellular; M, cell membrane; C, cytoplasm; N, nucleus. q FEBS 2001 MAPK mediates CTGF actions on chondrocytes (Eur. J. Biochem. 268) 6063 mediated by CTGF/Hcs24 in HCS-2/8 and RGC cells. The ERK pathway plays an important role in the proliferation of chondrocytes, while the p38 MAPK pathway plays a role in the differentiation of chondrocytes induced by CTGF/ Hcs24. Our findings suggest that two MAP kinase cascades mediate various important roles of CTGF/Hcs24 in the proliferation and differentiation of chondrocytes during endochondoral ossification. ACKNOWLEDGEMENTS This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science,Sports and Culture of Japan (to T.N., T.N., and M.T.), and grants from the Foundation for Growth Science in Japan (to M.T.), the Sumitomo Foundation (to M.T.) and the Research for the Future Programme of The Japan Society for the Promotion of Science (JSPS) (Project: Biological Tissue Engineering, JSPS-RFTF98100201). We thank Drs Satoshi Kubota, Takanori Eguchi, and Seiji Kondo for useful discussions, Drs Kumiko Nawachi and Norifumi Moritani for technical assistance, Dr Daisuke Ekuni for performing the Dunnett test, and Miss Yuki Nonami for secretarial assistance. REFERENCES 1. Bradham, D.M., Igarashi, A., Potter, R.L. & Grotendorst, G.R. (1991) Connective tissue growth factor: a cysteine-rich mitogen secreted by human vascular endothelial cells is related to the SRC- induced immediate early gene product CEF-10. J. Cell Biol. 114, 1285–1294. 2. Bork, P. (1993) The architecture of a new family of growth regulators related to connective tissue growth factor. FEBS Lett. 327, 125–130. 3. Lau, L.F. & Lam, S.C. (1999) The CCN family of angiogenic regulators: the integrin connection. Exp. Cell Res. 248, 44 –57. 4. Brigstock, D.R. (1999) The connective tissue growth factor/ cysteine-rich 61/nephroblastoma overexpressed (CCN) family. Endocr. Rev. 20, 189– 206. 5. Takigawa, M. (2000) Physiological roles of connective tissue growth factor (CTGF/Hcs24): promotion of endochondral ossifica- tion, angiogenesis and tissue remodeling. In Tissue Engineering for Therapeutic Use 4. (Ikada, Y. & Shimizu, Y., eds) pp. 1 –13. Elsevier, Amsterdam, the Netherlands. 6. Zhang, R., Averboukh, L., Zhu, W., Zhang, H., Jo, H., Dempsey, P.J., Coffey, R.J., Pardee, A.B. & Liang, P. (1998) Identification of rCop-1, a new member of the CCN protein family, as a negative regulator for cell transformation. Mol. Cell Biol. 18, 6131– 6141. 7. Hashimoto, Y., Shindo-Okada, N., Tani, M., Nagamachi, Y., Takeuchi, K., Shiroishi, T., Toma, H. & Yokota, J. (1998) Expression of the Elm1 gene, a novel gene of the CCN (connective tissue growth factor, Cyr61/Cef10, and neuroblastoma over- expressed gene) family, suppresses in vivo tumor growth and metastasis of K-1735 murine melanoma cells. J. Exp. Med. 187, 289– 296. 8. Hurvitz, J.R., Suwairi, W.M., Van Hul, W., El-Shanti, H., Superti- Furga, A., Roudier, J., Holderbaum, D., Pauli, R.M., Herd, J.K., Van Hul, E.V., et al. (1999) Mutations in the CCN gene family member WISP3 cause progressive pseudorheumatoid dysplasia. Nat. Genet. 23, 94–98. 9. Kumar, S., Hand, A.T., Connor, J.R., Dodds, R.A., Ryan, P.J., Trill, J.J., Fisher, S.M., Nuttall, M.E., Lipshutz, D.B., Zou, C., et al. (1999) Identification and cloning of a connective tissue growth factor-like cDNA from human osteoblasts encoding a novel regulator of osteoblast functions. J. Biol. Chem. 274, 17123– 17131. 10. Takigawa, M., Tajima, K., Pan, H.O., Enomoto, M., Kinoshita, A., Suzuki, F., Takano, Y. & Mori, Y. (1989) Establishment of a clonal human chondrosarcoma cell line with cartilage phenotypes. Cancer Res. 49, 3996 –4002. 11. Takigawa, M., Pan, H.O., Kinoshita, A., Tajima, K. & Takano, Y. (1991) Establishment from a human chondrosarcoma of a new immortal cell line with high tumorigenicity in vivo, which is able to form proteoglycan- rich cartilage-like nodules and to respond to insulin in vitro. Int. J. Cancer. 48, 717– 725. 12. Nakanishi, T., Kimura, Y., Tamura, T., Ichikawa, H., Yamaai, Y., Sugimoto, T. & Takigawa, M. (1997) Cloning of a mRNA prefer- entially expressed in chondrocytes by differential display-PCR from a human chondrocytic cell line that is identical with connective tissue growth factor (CTGF )mRNA.Biochem. Biophys. Res. Commun. 234, 206–210. 13. Nakanishi, T., Nishida, T., Shimo, T., Kobayashi, K., Kubo, T., Tamatani, T., Tezuka, K. & Takigawa, M. (2000) Effects of CTGF/ Hcs24, a product of a hypertrophic chondrocyte-specific gene, on the proliferation and differentiation of chondrocytes in culture. Endocrinology 141, 264– 273. 14. Nishida, T., Nakanishi, T., Shimo, T., Asano, M., Hattori, T., Tamatani, T., Tezuka, K. & Takigawa, M. (1998) Demonstration of receptors specific for connective tissue growth factor on a human chondrocytic cell line (HCS-2/8). Biochem. Biophys. Res. Commun. 247, 905–909. 15. Nishida, T., Nakanishi, T., Asano, M., Shimo, T. & Takigawa, M. (2000) Effects of CTGF/Hcs24, a hypertrophic chondrocyte- specific gene product, on the proliferation and differentiation of osteoblastic cells in vitro. J. Cell. Physiol. 184, 197–206. 16. Igarashi, A., Okochi, H., Bradham, D.M. & Grotendorst, G.R. (1993) Regulation of connective tissue growth factor gene expression in human skin fibroblasts and during wound repair. Mol. Biol. Cell. 4, 637– 645. 17. Shimo, T., Nakanishi, T., Kimura, Y., Nishida, T., Ishizeki, K., Matsumura, T. & Takigawa, M. (1998) Inhibition of endogenous expression of connective tissue growth factor by its antisense oligonucleotide and antisense RNA suppresses proliferation and migration of vascular endothelial cells. J. Biochem. 124, 130 –140. 18. Shimo, T., Nakanishi, T., Nishida, T., Asano, M., Kanyama, M., Kuboki, T., Tamatani, T., Tezuka, K., Takemura, M., Matsumura, T. & Takigawa, M. (1999) Connective tissue growth factor induces the proliferation, migration, and tube formation of vascular endothelial cells in vitro, and angiogenesis in vivo. J. Biochem. 126, 137–145. 19. Hishikawa, K., Oemar, B.S., Tanner, F.C., Nakaki, T., Luscher, T.F. & Fujii, T. (1999) Connective tissue growth factor induces apoptosis in human breast cancer cell line MCF-7. J. Biol. Chem. 274, 37461–37466. 20. Babic, A.M., Chen, C.C. & Lau, L.F. (1999) Fisp12/mouse connective tissue growth factor mediates endothelial cell adhesion and migration through integrin alphavbeta3, promotes endothelial cell survival, and induces angiogenesis in vivo. Mol. Cell Biol. 19, 2958–2966. 21. Jedsadayanmata, A., Chen, C.C., Kireeva, M.L., Lau, L.F. & Lam, S.C. (1999) Activation-dependent adhesion of human platelets to Cyr61 and Fisp12/mouse connective tissue growth factor is mediated through integrin alpha (IIb) beta (3). J. Biol. Chem. 274, 24321–24327. 22. Chen, C.C., Chen, N. & Lau, L.F. (2000) The angiogenic factors Cyr61 and CTGF induce adhesive signaling in primary human skin fibroblasts. J. Biol. Chem. 18, 18. 23. Kubota, S., Kondo, S., Eguchi, T., Hattori, T., Nakanishi, T., Pomerantz, R.J. & Takigawa, M. (2000) Identification of an RNA element that confers post-transcriptional repression of connective tissue growth factor/hypertrophic chondrocyte specific 24 (ctgf/ hcs24 ) gene: similarities to retroviral RNA–protein interactions. Oncogene 19, 4773–4786. 24. Kubota, S., Hattori, T., Shimo, T., Nakanishi, T. & Takigawa, T. 6064 G. Yosimichi et al. (Eur. J. Biochem. 268) q FEBS 2001 (2000) Novel intracellular effects of human connective tissue growth factor expressed in COS-7 cells. FEBS Lett. 474, 58– 62. 25. Yoon, Y.M., Oh, C.D., Kim, D.Y., Lee, Y.S., Park, J.W., Huh, T.L., Kang, S.S. & Chun, J.S. (2000) Epidermal growth factor negatively regulates chondrogenesis of mesenchymal cells by modulating the protein kinase C-alpha, Erk-1, and p38 MAPK signaling pathways. J. Biol. Chem. 275, 12353–12359. 26. Pang, L., Sawada, T., Decker, S.J. & Saltiel, A.R. (1995) Inhibition of MAP kinase kinase blocks the differentiation of PC-12 cells induced by nerve growth factor. J. Biol. Chem. 270, 13585–13588. 27. Murakami, S., Kan, M., McKeehan, W.L. & de Crombrugghe, B. (2000) Up-regulation of the chondrogenic Sox9 gene by fibroblast growth factors is mediated by the mitogen-activated protein kinase pathway. Proc. Natl Acad. Sci. USA 97, 1113– 1118. 28. Yonekura, A., Osaki, M., Hirota, Y., Tsukazaki, T., Miyazaki, Y., Matsumoto, T., Ohtsuru, A., Namba, H., Shindo, H. & Yamashita, S. (1999) Transforming growth factor-beta stimulates articular chondrocyte cell growth through p44/42 MAP kinase (ERK) activation. Endocr. J. 46, 545– 553. 29. Hirota, Y., Tsukazaki, T., Yonekura, A., Miyazaki, Y., Osaki, M., Shindo, H. & Yamashita, S. (2000) Activation of specific MEK- ERK cascade is necessary for TGF beta signaling and crosstalk with PKA and PKC pathways in cultured rat articular chondrocytes. Osteoarthritis Cartilage 8, 241– 247. 30. Rao, V.N. & Reddy, E.S. (1994) elk-1 proteins interact with MAP kinases. Oncogene 9, 1855–1860. 31. Jiang, Y., Chen, C., Li, Z., Guo, W., Gegner, J.A., Lin, S. & Han, J. (1996) Characterization of the structure and function of a new mitogen- activated protein kinase (p38beta). J. Biol. Chem. 271, 17920–17926. 32. Davis, R.J. (2000) Signal transduction by the JNK group of MAP kinases. Cell 103, 239–252. 33. Hill, C.S. & Treisman, R. (1995) Transcriptional regulation by extracellular signals: mechanisms and specificity. Cell 80, 199– 211. 34. Moriguchi, T., Kuriyanagi, N., Yamaguchi, K., Gotchi, Y., Irie, K., Kano, T., Shirakabe, K., Muro, Y., Shibuya, H., Matsumoto, K., et al. (1996) A novel kinase cascade madiated by mitogen-activated protein kinase kinase 6 and MKK3. J. Biol. Chem. 271, 13675–13679. 35. Alessi, D.R., Cuenda, A., Cohen, P., Dudley, D.T. & Saltiel, A.R. (1995) PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J. Biol. Chem. 270, 27489–27494. 36. Dudley, D.T., Pang, L., Decker, S.J., Bridges, A.J. & Saltiel, A.R. (1995) A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc. Natl Acad. Sci. USA 92, 7686–7689. 37. Cuenda, A., Rouse, J., Doza, Y.N., Meier, R., Cohen, P., Gallagher, T.F., Young, P.R. & Lee, J.C. (1995) SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1. FEBS Lett. 364, 229– 233. 38. Kawamura, H., Otsuka, T., Matsuno, H., Niwa, M., Matsui, N., Kato, K., Uematsu, T. & Kozawa, O. (1999) Endothelin-1 stimulates heat shock protein 27 induction in osteoblasts: involve- ment of p38 MAP kinase. Am. J. Physiol. 277, E1046– E1054. 39. Tamura, T., Nakanishi, T., Kimura, Y., Hattori, T., Sasaki, K., Norimatsu, H., Takahashi, K. & Takigawa, M. (1996) Nitric oxide mediates interleukin-1-induced matrix degradation and basic fibroblast growth factor release in cultured rabbit articular chondrocytes: a possible mechanism of pathological neovascular- ization in arthritis. Endocrinology 137, 3729 –3737. 40. Sasaki, K., Hattori, T., Fujisawa, T., Takahashi, K., Inoue, H. & Takigawa, M. (1998) Nitric oxide mediates interleukin-1-induced gene expression of matrix metalloproteinases and basic fibroblast growth factor in cultured rabbit articular chondrocytes. J. Biochem. 123, 431–439. 41. Zhen, X., Wei, L., Wu, Q., Zhang, Y. & Chen, Q. (2000) Mitogen- activated protein kinase p38 mediates regulation of chondrocyte differentiation by parathyroid hormone. J. Biol. Chem. 29, 29. 42. Nakamura, K., Shirai, T., Morishita, S., Uchida, S., Saeki-Miura, K. & Makishima, F. (1999) p38 mitogen-activated protein kinase functionally contributes to chondrogenesis induced by growth/ differentiation factor-5 in ATDC5 cells. Exp. Cell Res. 250, 351– 363. 43. Liu, C., Liu, X.J., Crowe, P.D., Kelner, G.S., Fan, J., Barry, G., Manu, F., Ling, N., De Souza, E.B. & Maki, R.A. (1999) Nephro- blastoma overexpressed gene (NOV) codes for a growth factor that induces protein tyrosine phosphorylation. Gene 238, 471– 478. 44. Miwa, M., Kozawa, O., Tokuda, H. & Uematsu, T. (1999) Mitogen- activated protein (MAP) kinases are involved in interleukin-1 (IL-1)-induced IL-6 synthesis in osteoblasts: modulation not of p38 MAP kinase, but of p42/p44 MAP kinase by IL-1-activated protein kinase C. Endocrinology 140, 5120–5125. 45. De ´ rijard, B., Raingeaud, J., Barrett, T., Wu, I.H., Han, J., Ulevitch, R.J., Davis, R.J. (1995) Independent human MAP kinase signal transduction pathways defined by MEK and MKK isoforms. Science 267, 682 –685. 46. Enslen, H., Raineaud, J. & Davis, R.J. (1998) Selective activation of p38 mitogen-activated protein (MAP) kinase isoforms by the MAP kinase kinase MKK3 and MKK6. J. Biol. Chem. 273, 1741–1748. q FEBS 2001 MAPK mediates CTGF actions on chondrocytes (Eur. J. Biochem. 268) 6065 . CTGF/Hcs24 induces chondrocyte differentiation through a p38 mitogen-activated protein kinase (p38MAPK), and proliferation through a p44/42 MAPK/extracellular-signal. MAPK/extracellular-signal regulated kinase (ERK) Gen Yosimichi 1,2 , Tohru Nakanishi 1 , Takashi Nishida 1,3 , Takako Hattori 1 , Teruko Takano-Yamamoto 2 and Masaharu