Báo cáo khoa học: Cartducin stimulates mesenchymal chondroprogenitor cell proliferation through both extracellular signal-regulated kinase and phosphatidylinositol 3-kinase⁄Akt pathways pot

Cartducin stimulates mesenchymal chondroprogenitor cell proliferation through both extracellular signal-regulated kinase and phosphatidylinositol 3-kinase⁄ Akt pathways Hironori Akiyama 1,2 , Souhei Furukawa 2 , Satoshi Wakisaka 1 and Takashi Maeda 1 1 Department of Anatomy and Cell Biology, Graduate School of Dentistry, Osaka University, Japan 2 Department of Radiology, Graduate School of Dentistry, Osaka University, Japan Cartilage formation is driven by mesenchymal chon- droprogenitor cells that proliferate and differentiate into chondrocytes. The molecular mechanisms by which growth factors regulate the fate of these cells are not well defined. We recently discovered a secre- tory protein, cartducin, which is an  27 kDa polypep- tide containing an N-terminal signal peptide, a short variable domain, a collagen-like domain, and a C-ter- minal C1q-like globular domain. Its mRNA is predominantly and highly expressed in developing cartilages [1]. Interestingly, cartducin has structural homology with a 30 kDa serum protein Acrp30 ⁄ adipo- nectin, which is exclusively and highly expressed in differentiated adipocytes [2], and has been shown to be dysregulated in various forms of obesity in mice and humans [3,4]. Because cartducin is a paralog of Acrp30 ⁄ adiponectin, cartducin is a novel member of a newly designated C1q family of proteins [5,6]. The Keywords cartducin; chondroprogenitor cells; MAPK; PI3K ⁄ Akt; proliferation Correspondence T. Maeda, Department of Anatomy and Cell Biology, Graduate School of Dentistry, Osaka University, 1–8 Yamadaoka, Suita, Osaka 565–0871, Japan Fax: +81 6 6879 2875 Tel: +81 6 6879 2874 E-mail: tmaeda@dent.osaka-u.ac.jp (Received 20 November 2005, revised 20 February 2006, accepted 20 March 2006) doi:10.1111/j.1742-4658.2006.05240.x Cartducin, a paralog of Acrp30 ⁄ adiponectin, is a secretory protein pro- duced by both chondrogenic precursors and proliferating chondrocytes, and belongs to a novel C1q family of proteins. We have recently shown that cartducin promotes the growth of both mesenchymal chondroprogeni- tor cells and chondrosarcoma-derived chondrocytic cells in vitro. However, the cartducin-signaling pathways responsible for the regulation of cell pro- liferation have not been documented. In this study, we examined whether cartducin exists in serum and further investigated the intracellular signaling pathways stimulated by cartducin in mesenchymal chondroprogenitor cells. Western blot analysis showed that, unlike Acrp30⁄ adiponectin, cartducin was undetectable in mouse serum. Next, mesenchymal chondroprogenitor N1511 cells were stimulated with cartducin, and three major groups of mitogen-activated protein kinase (MAPK) pathways and the phosphatidyl- inositol 3-kinase (PI3K) ⁄ Akt signaling pathway were examined. Cartducin activated extracellular signal-regulated kinase 1 ⁄ 2 (ERK1 ⁄ 2) and Akt, but not c-jun N-terminal kinase (JNK) nor p38 MAPK. The MEK1 ⁄ 2 inhib- itor, U0126, blocked cartducin-stimulated ERK1 ⁄ 2 phosphorylation and suppressed the DNA synthesis induced by cartducin in N1511 cells. The PI3K inhibitor, LY294002, blocked cartducin-stimulated Akt phosphoryla- tion and a decrease in cartducin-induced DNA synthesis in N1511 cells was also observed. These data suggest that cartducin is a peripheral skeletal growth factor, and that the proliferation of mesenchymal chondroprogeni- tor cells stimulated by cartducin is associated with activations of the ERK1 ⁄ 2 and PI3K ⁄ Akt signaling pathways. Abbreviations BrdU, bromodeoxyuridine; ERK, extracellular signal-regulated kinase; JNK, c-jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MEK, MAPK ⁄ ERK kinase; a-MEM, alpha-minimal essential medium; PI3K, phosphatidylinositol 3-kinase. FEBS Journal 273 (2006) 2257–2263 ª 2006 The Authors Journal compilation ª 2006 FEBS 2257 members of this family contain a C-terminal C1q-like globular domain and are involved in processes as diverse as host defense, inflammation, apoptosis, auto- immunity, cell differentiation, organogenesis, hiberna- tion and insulin-resistant obesity. More recently, we found that cartducin is strongly expressed in both the chondrogenic cell lineage in the sclerotome and proliferating chondrocytes in the growth plate of cartilage, and it promotes the prolifer- ation of mesenchymal chondroprogenitor cells and chondrocytes in vitro [7]. Although cartducin is a bio- logically active molecule involved in both embryonic cartilage development and postnatal longitudinal bone growth, the signaling pathways activated upon cartdu- cin stimulation have not been documented. Because a high mitogenic activity of mesenchymal chondropro- genitor cells is initially required to produce enough cells for the process of chondrogenesis [8], elucidation of the signaling pathways responsible for cartducin- induced DNA synthesis by mesenchymal chondropro- genitor cells is necessary to more fully understand the molecular mechanisms of cartilage formation. Mitogen-activated protein kinase (MAPK) pathways are essential mitogenic pathways in many cell lines and are responsible for various growth factors. Down- stream of these pathways can simply be classified into three major groups mediated by the following kinases: extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK [9,10]. Another signaling pathway associated with the control of mitogenesis is the phosphatidylinositol 3-kinase (PI3K) ⁄ Akt pathway. It is also an important mediator of cell growth and survival in response to growth factors and other signals. PI3K activates Akt serine ⁄ threonine kinase by generating specific inositol phos- pholipids, which recruit Akt to the cell membrane and enable its activation [11]. In this study, we examined the possibility that cartducin is a serum protein and further investigated the possible signaling pathways activated by cartducin in the regulation of DNA synthesis using the mouse mesenchymal chondroprogenitor cell line, N1511 [12]. The results show that cartducin, a local reg- ulatory factor, exerts its effects on the proliferation of mesenchymal chondroprogenitor cells by activating both the ERK1 ⁄ 2 and PI3K ⁄ Akt pathways. Results Cartducin is a local regulatory factor but not a systemic hormone Cartducin is a novel growth factor which belongs to a newly designated family of proteins, the C1q ⁄ TNF superfamily, all of which contain a C-terminal C1q-like globular domain and most of which contain a colla- genous region. Based on the structural similarities of cartducin to the Acrp30 ⁄ adiponectin, which has been shown to play important roles in the regulation of glu- cose and lipid metabolism as a hormone, we investi- gated the possibility that cartducin is also a serum protein. A protein of identical molecular mass to cart- ducin can be detected in the total tissue protein extrac- ted from mouse growth plate cartilage. In contrast, unlike Acrp30 ⁄ adiponectin, we failed to detect cartdu- cin in mouse serum using western blot analysis (Fig. 1). These results suggest that cartducin exerts its effects in vivo in an autocrine ⁄ paracrine manner. Cartducin activates the ERK1 ⁄ 2 pathway in mesenchymal chondroprogenitor N1511 cells Cartducin is known to play important roles in regula- ting both chondrogenesis and cartilage development by its direct stimulatory action on the proliferation of chondrogenic precursors and chondrocytes. MAPK pathways are essential mitogenic pathways in many cells and are responsible for various growth factors. Three major groups of MAPKs, such as ERK, JNK, and p38 MAPK, have been identified. Therefore, we analyzed the effects of cartducin on the phosphoryla- tion of these three groups of MAPKs in N1511 cells. Western blot analysis showed that increased ERK1 ⁄ 2 phosphorylation in N1511 cells treated with 10 lgÆmL )1 of cartducin was detected after 5 min, with maximal increase occurring after 15 min of treatment and decreasing after 1 h (Fig. 2A). A dose-dependent increase in ERK1 ⁄ 2 phosphorylation in N1511 cells after 30 min of treatment was detected at 2, 5, and 10 lgÆmL )1 of cartducin (Fig. 2B). In contrast, cartdu- cin had no effect on the activities of JNK1 ⁄ 2 and p38 37 25 Cartducin kDa Cartilage Serum Fig. 1. Cartducin is not a serum protein. One microliter of mouse serum was boiled for 5 min in 2 · sample buffer and analyzed by SDS ⁄ PAGE and western blot using anti-mouse IgG. Antibody was visualized with an anti-goat IgG coupled to alkaline phosphatase. A protein of identical molecular mass to cartducin can be detected in the total tissue protein extracted from mouse growth plate carti- lage. However, cartducin could not be detected in serum from mice. Cartducin activates MAPK and PI3K ⁄ Akt H. Akiyama et al. 2258 FEBS Journal 273 (2006) 2257–2263 ª 2006 The Authors Journal compilation ª 2006 FEBS MAPK, and none of their phosphorylated forms was detected (Fig. 3). Both ERK1 and ERK2 have been shown to be activated by their upstream activators, MEK1 and MEK2. We next examined the action of the specific MEK1 ⁄ 2 inhibitor, U0126, on ERK1 ⁄ 2 pathway activation. Phosphorylation of ERK1 ⁄ 2 induced by cartducin was completely inhibited by pre- treatment with U0126 (10 lm) (Fig. 4). ERK1 ⁄ 2 pathway activation is involved in cartducin-induced proliferation of mesenchymal chondroprogenitor N1511 cells Because western blot analysis confirmed cartducin- induced ERK1 ⁄ 2 pathway activation in N1511 cells, we next determined whether cartducin-induced mesen- chymal chondroprogenitor cell proliferation is medi- ated through activation of the ERK1 ⁄ 2 pathway. N1511 cells were pretreated with the MEK1 ⁄ 2 inhib- itor U0126 (10 lm), JNK inhibitor SP600125 (20 lm), or p38 MAPK inhibitor SB203580 (10 lm) for 1 h before and for the duration of the stimulation, and DNA synthesis analysis by estimating the incorpor- ation of bromodeoxyuridine (BrdU) was performed (Fig. 5). Cartducin induced about a twofold increase in DNA synthesis in N1511 cells compared with the buffer-treated control culture. Treatment with U0126, SP600125, or SB203580 suppressed the basal level of DNA synthesis in N1511 cells compared with the dimethylsulfoxide-treated control culture, respectively. Fig. 3. Effects of cartducin on phosphorylation of JNK and p38 MAPK in N1511 cells. Mesenchymal chondroprogenitor N1511 cells stimulated with cartducin (10 lgÆmL )1 ) for 5, 15, 30, and 60 min were analyzed by western blot for phosphorylated JNK1 ⁄ 2 (p- JNK1 ⁄ 2), total JNK1 ⁄ 2, phosphorylated p38 MAPK (p-p38 MAPK), and total p38 MAPK. Cartducin had no effect on the phosphoryla- tion of JNK1 ⁄ 2 and p38 MAPK, and none of their phosphorylated forms were detected. Cartducin: – –+ ++–– + U0126: ERK1/2 p-ERK1/2 Fig. 4. Effects of MAPK inhibitor on phosphorylation of ERK in N1511 cells. Mesenchymal chondroprogenitor N1511 cells were pretreated with MEK1 ⁄ 2 inhibitor, U0126 (10 l M), for 1 h, stimula- ted with cartducin (10 lgÆmL )1 ) for 15 min, and then analyzed by western blot for phosphorylated ERK1 ⁄ 2 (p-ERK1 ⁄ 2). Treatment with U0126 inhibited cartducin-induced ERK1 ⁄ 2 phosphorylation. A B Fig. 2. Effects of cartducin on phosphorylation of ERK in mesen- chymal chondroprogenitor N1511 cells. (A) Subconfluent cultures of N1511 cells stimulated with cartducin (10 lgÆmL )1 ) for 5, 15, 30, and 60 min were analyzed by western blot for phosphorylated ERK1 ⁄ 2 (p-ERK1 ⁄ 2) and total ERK1 ⁄ 2. Cartducin increased the p-ERK1 ⁄ 2 level after 5 min incubation. Peak activation of ERK1 ⁄ 2 occurred at 15 min. (B) Cells were stimulated with increasing doses of cartducin (2–10 lgÆmL )1 ) for 30 min. Cartducin induced a dose- dependent activation of ERK1 ⁄ 2. Fig. 5. Effects of MAPK inhibitors on DNA synthesis in mesenchy- mal chondroprogenitor N1511 cells. Subconfluent cultures of N1511 cells were serum starved, preincubated with MEK1 ⁄ 2 inhib- itor U0126 (10 l M), JNK inhibitor SP600125 (20 lM), or p38 MAPK inhibitor SB203580 (10 l M) for 1 h before cartducin treatment (8 lgÆmL )1 ), and bromodeoxyuridine (BrdU) incorporation was measured. Data are represented as the mean ± SD of triplicate determinations. P < 0.05 versus cartducin-untreated each control. Similar results were obtained from two independent experiments. H. Akiyama et al. Cartducin activates MAPK and PI3K ⁄ Akt FEBS Journal 273 (2006) 2257–2263 ª 2006 The Authors Journal compilation ª 2006 FEBS 2259 Inhibition of the ERK1 ⁄ 2 pathway by pretreatment with U0126 led to a block of cartducin-induced N1511 cell proliferation. By contrast, inhibition of JNK or p38 MAPK pathways by SP600125 or SB203580 did not affect cartducin-induced N1511 cell proliferation. Cartducin activates the PI3K ⁄ Akt pathway in N1511 cells The PI3K ⁄ Akt pathway is also an important mediator of cell growth and survival in response to growth factors and other signals. We therefore analyzed the effects of cartducin on activation of the PI3K ⁄ Akt pathway in mesenchymal chondroprogenitor N1511 cells. Western blot analysis showed that increased Akt phosphorylation in N1511 cells treated with 10 lgÆmL )1 of cartducin was detected after 5 min, with maximal increase occurring after 30 min of treatment and decreasing after 1 h (Fig. 6A). A dose-dependent increase in Akt phosphorylation in N1511 cells after 30 min of treatment was detected at 2, 5, and 10 lgÆmL )1 of cartducin (Fig. 6B). Next, the action of the specific PI3K inhibitor, LY294002, on Akt phos- phorylation was examined. Phosphorylation of Akt induced by cartducin was inhibited completely by pre- treatment with LY294002 (25 lm) (Fig. 6C). PI3K ⁄ Akt pathway activation is involved in cartducin-induced proliferation of mesenchymal chondroprogenitor N1511 cells Because western blot analysis confirmed cartducin- induced PI3K ⁄ Akt pathway activation in N1511 cells, we further determined whether cartducin-induced mesenchymal chondroprogenitor cell proliferation is mediated through activation of the PI3K ⁄ Akt path- way. N1511 cells were pretreated with the PI3K inhib- itor, LY294002 (1–25 lm), for 1 h before and for the duration of the stimulation, and DNA synthesis analy- sis was performed (Fig. 7). Similar to treatments with MAPK inhibitors, treatment with LY294002 also sup- pressed the basal level of DNA synthesis in N1511 cells. Inhibition of the PI3K ⁄ Akt pathway by pretreat- ment with LY294002 significantly reduced DNA syn- thesis in N1511 cells in a dose-dependent manner after cartducin stimulation, suggesting involvement of the PI3K ⁄ Akt pathway in the ability of cartducin to sti- mulate proliferation of these cells. Discussion Skeletal development requires the condensation of multipotential mesenchymal cells to differentiate A B C Fig. 6. Cartducin-induced Akt phosphorylation is dependent on PI3K activity. (A) Time course of cartducin-induced phosphorylation of Akt in N1511 mesenchymal chondroprogenitor cells. The cells stimula- ted with cartducin (10 lgÆmL )1 ) for 5, 15, 30, and 60 min were ana- lyzed by western blot for phosphorylated Akt (p-Akt) and total Akt. Cartducin increased the p-Akt level after 5 min incubation. Peak acti- vation of Akt occurred at 30 min. (B) Dose–response of cartducin- induced phosphorylation of Akt in N1511 cells. The cells were stimulated with increasing doses of cartducin (2–10 lgÆmL )1 ) for 30 min. Cartducin induced a dose-dependent activation of Akt. (C) Effects of PI3K inhibitor on phosphorylation of Akt in N1511 cells. Cells were pretreated with the PI3K inhibitor, LY294002 (25 l M), for 1 h, stimulated with cartducin (10 lgÆmL )1 ) for 30 min, and then analyzed by western blot for phosphorylated Akt (p-Akt). Treatment with LY294002 inhibited cartducin-induced Akt phosphorylation. Fig. 7. Effects of PI3K inhibitors on DNA synthesis in mesenchymal chondroprogenitor N1511 cells. Subconfluent cultures of N1511 cells were serum starved, preincubated with PI3K inhibitor, LY294002 (1–25 l M), for 1 h before cartducin treatment (8 lgÆmL )1 ), and BrdU incorporation was measured. Treatment of N1511 cells with LY294002 inhibited the cartducin-stimulated DNA synthesis in a concentration-dependent manner. Data are represen- ted as the mean ± SD of triplicate determinations. P < 0.05 versus cartducin-untreated control. Similar results were obtained from two independent experiments. Cartducin activates MAPK and PI3K ⁄ Akt H. Akiyama et al. 2260 FEBS Journal 273 (2006) 2257–2263 ª 2006 The Authors Journal compilation ª 2006 FEBS toward the various cell types. One such process is cartilage formation from undifferentiated mesenchy- mal cells. Discoveries of diffusible factors that act on the chondrogenic cell lineage and ⁄ or chondrocytes have dramatically improved our understanding of the molecular mechanisms of skeletal development. Cartducin is a recently described cartilage-derived secretory protein [1], and it has been indicated to play important roles in regulating both embryonic cartilage development and postnatal longitudinal bone growth by directly promoting the proliferation of mesenchymal chondroprogenitor cells and chond- rocytes [7]. Because cartducin shares a similar modu- lar organization to the serum protein Acrp30 ⁄ adiponectin, which has been shown to play import- ant roles as a hormone in the regulation of glucose and lipid metabolism, we considered the possibility that cartducin might be not only a local regulatory factor, but also a systemic hormone. However, we could not detect cartducin in 1 lL (Fig. 1) or more (data not shown) of mouse serum using western blot analysis. These data suggested that cartducin is not a serum protein, and thus cartducin may exert its stimulatory action on the proliferation of chondro- genic precursors and chondrocytes in vivo in an autocrine ⁄ paracrine fashion only. The expression of cartducin starts in the sclero- tome, which contains a chondrogenic cell lineage during mouse embryogenesis. Recombinant cartducin induced DNA synthesis in mesenchymal chondropro- genitor N1511 cells in a dose-dependent manner [7]. In contrast to Acrp30 ⁄ adiponectin [13], a cartducin- specific receptor has not yet been identified and cloned, and little is known about its signaling path- ways. To gain insight into the mechanisms by which cartducin promotes proliferation of mesenchymal chondroprogenitor cells, we evaluated the signaling events. MAPKs are well known to play an essential role in controlling cell proliferation and differentia- tion [9,10]. Indeed, the ERK1 ⁄ 2 pathway is involved in controlling mesenchymal chondroprogenitor cell proliferation [14]. In this study, we found that cart- ducin induces activation of ERK1 ⁄ 2 in mesenchymal chondroprogenitor N1511 cells but has no effect on activation of JNK and p38 MAPK. Next, we investi- gated the effects of MAPK inhibitors on the prolif- eration of cartducin-stimulated N1511 cells by analyzing DNA synthesis. Inhibition of the ERK1 ⁄ 2 signaling pathway by the specific MEK1 ⁄ 2 inhib- itor, U0126, but not the specific JNK inhibitor, SP600125, nor the specific p38 MAPK inhibitor, SB203580, blocked cartducin-stimulated proliferation of N1511 cells. The data suggest that the ERK1 ⁄ 2 pathway has an important role in the proliferation of mesenchymal chondroprogenitor cells stimulated by cartducin. JNK or p38 MAPK pathways do not seem to be involved in the cartducin-stimulated pro- liferation of these cells. Another signaling pathway associated with the con- trol of cell proliferation is the PI3K ⁄ Akt pathway. It is also an important mediator of cell growth and survival in response to growth factors and other signals. PI3K activates Akt serine ⁄ threonine kinase by generating specific inositol phospholipids, which recruit Akt to the cell membrane and enable its activation [11]. Akt mediates cell survival and growth signals by phos- phorylating and inactivating proapoptic proteins [15]. Moreover, growth retardation has been reported in knockout mice for Akt1 gene, which demonstrates that Akt is important for normal growth [16]. In the pre- sent study, we also found that the PI3K ⁄ Akt pathway was activated by cartducin stimulation, and inhibition of this signaling pathway by a specific PI3K inhibitor, LY294002, blocked the cartducin-stimulated pro- liferation of N1511 cells. The data suggest that the PI3K ⁄ Akt pathway is also involved in cartducin-stimu- lated proliferation of mesenchymal chondroprogenitor cells. Our results indicated that cartducin initiates MAPK and PI3K signaling cascades in mesenchymal chondroprogenitor cells, leading to the phosphoryla- tion of ERK1 ⁄ 2 and Akt, respectively, and both pathways are required for the cells to complete pro- gression through the mitotic cycle. This finding is consistent with the well-established roles of these signaling pathways in the propagation of mitogenic signals [9,15]. Although Akt has been thought to play a major role, mainly in cell survival, recent data suggest that PI3K activation is required for the pro- gression of mitosis, promoting the entry of quiescent cells into the S phase, and the downstream phos- phorylation of Akt is in part responsible for the propagation of this signal [17]. In particular, the mitogenic response to platelet-derived growth factor involves the sequential activation of the MAPK and PI3K pathways, and these different phases of signa- ling are a general requirement in mitogenic signa- ling [18]. The inhibition of the mitogenic response to cartducin by the specific PI3K inhibitor, LY294002, is consistent with this role for PI3K in mitogenesis. In conclusion, we have demonstrated that cartdu- cin, a novel C1q family member, is a peripheral skel- etal growth factor, and mitogenic response to cartducin by mesenchymal chondroprogenitor cells requires the activation of both the ERK1 ⁄ 2 and PI3K ⁄ Akt pathways. H. Akiyama et al. Cartducin activates MAPK and PI3K ⁄ Akt FEBS Journal 273 (2006) 2257–2263 ª 2006 The Authors Journal compilation ª 2006 FEBS 2261 Experimental procedures Reagents Mouse recombinant cartducin was prepared as described [7]. Anti-mouse cartducin (also called CORS26) serum was purchased from R&D Systems (Minneapolis, MN). Anti- (ERK1 ⁄ 2), anti-(JNK1 ⁄ 2), and anti-(p38 MAPK) sera were purchased from Sigma-Aldrich (St. Louis, MO). Anti- (p-ERK1 ⁄ 2), anti-(p-JNK1 ⁄ 2), anti-(p-p38 MAPK), anti- Akt, and anti-(p-Akt) sera were purchased from Cell Signaling Technology, Inc. (Beverly, MA). MEK1 ⁄ 2 inhi- bitor U0126, JNK inhibitor SP600125, p38 MAPK inhi- bitor SB203580, and PI3K inhibitor LY294002 were purchased from Calbiochem (San Diego, CA). Cell lines and cell culture The mouse mesenchymal chondroprogenitor cell line N1511 [12] was cultured in alpha-minimal essential med- ium (a-MEM; Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; PAA Laboratories, Linz, Aus- tria), 2 mml-glutamine, and penicillin ⁄ streptomycin at 37 °C in a humidified atmosphere containing 5% CO 2 . To investigate the effects of cartducin on the MAPK or PI3K ⁄ Akt signaling pathways, cells were seeded at a den- sity of 1 · 10 4 cells per well in 24-well plates and grown for 48 h. The cells were then washed and cultured for 24 h in the medium without serum. Subsequently, 10 lgÆmL )1 of recombinant cartducin was added to the medium for 5, 15, 30, and 60 min. In another set of experiments, cells were treated with an increasing doses of recombinant cartducin (2–10 lgÆmL )1 ) for 30 min. For experiments with protein kinase inhibitors, cells were pre- treated with specific inhibitors for 1 h prior to cartducin treatment. Western blot analysis Total cellular protein was prepared by lysing cells in Cel- Lytic lysis buffer (Sigma-Aldrich) containing protease inhibitor (Sigma-Aldrich) and phosphatase inhibitor (Sigma-Aldrich) cocktails. Total tissue protein was extrac- ted from rib growth plate cartilage from 10-day-old mice. The proteins (20 lg) were separated by SDS ⁄ PAGE (10% polyacrylamide gel) and transferred to a poly(vinylidene difluoride) membrane (Bio-Rad, Hercules, CA) as described previously [19]. Membranes were blocked for 30 min at room temperature and then incubated with anti p-(ERK1 ⁄ 2), anti-(p-JNK1 ⁄ 2), anti-(p-p38 MAPK), or anti-(p-Akt) sera for 18 h at 4 °C. In other cases, the membranes were incubated with first antibodies for 1 h at room temperature. The detection of bound antibodies was performed by the WesternBreeze Chromogenic Detect- ion System (Invitrogen, Carlsbad, CA) using alkaline phosphatase-conjugated donkey anti-goat IgG, or anti- rabbit IgG serum (Promega, Madison WI). Measurement of DNA synthesis To determine the growth-stimulatory effect of cartducin on mesenchymal chondroprogenitor cells, a BrdU assay was performed as described previously [7]. In brief, N1511 cells were seeded at a density of 1 · 10 4 cells per well in 96-well plates and grown for 24 h. The cells were then washed and the medium was replaced with a-MEM containing 0.3% FBS for 24 h. Subsequently, 8 lgÆmL )1 of recombinant cartducin was added to the medium, incubated for 24 h, and labeled with BrdU for the last 3 h of incubation. To determine the effects of inhibitors on the cartducin-induced proliferation of N1511 cells, cells were treated with different inhibitors for 1 h before and for the duration of the stimu- lation with cartducin. In the control experiments, 50 mm NaH 2 PO 4 (pH 8.0) containing 1 mm EDTA and ⁄ or dimeth- ylsulfoxide was added to the culture. Statistical analysis An unpaired Student’s t-test was used for statistical analysis of the experiments. Error bars represent SD, and P < 0.05 was taken as the level of significance. Acknowledgements This work was supported by Grants-in-aid for Scienti- fic Research (No. 16791142) and the 21st Century Center of Excellence Program from the Ministry of Education, Culture, Sports, Science and Technology of Japan. 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