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p70 S6 kinase limits tumor necrosis factor-α-induced interleukin-6 synthesis in osteoblast-like cells
Molecular and Cellular Endocrinology 315 (2010) 195–200
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p70 S6 kinase limits tumor necrosis factor-␣-induced interleukin-6 synthesis in osteoblast-like cells Chiho Minamitani a,b , Haruhiko Tokuda b,c , Seiji Adachi b , Rie Matsushima-Nishiwaki b , Junichi Yamauchi b , Kenji Kato a,b , Hideo Natsume a,b , Jun Mizutani a , Osamu Kozawa b,∗ , Takanobu Otsuka a a
Department of Orthopedic Surgery, Nagoya City University Graduate School of Medical Sciences, Nagoya 467-8601, Japan Department of Pharmacology, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu 501-1194, Japan c Department of Clinical Laboratory, National Hospital for Geriatric Medicine, National Center for Geriatrics and Gerontology, Obu 474-8511, Japan b
a r t i c l e
i n f o
Article history: Received 11 June 2009 Received in revised form 7 October 2009 Accepted 16 October 2009 Keywords: TNF-␣ p70 S6 kinase IL-6 Osteoblast
a b s t r a c t Our previous study demonstrated that tumor necrosis factor-␣ (TNF-␣) stimulates the synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, via p44/p42 mitogen-activated protein (MAP) kinase and phosphatidylinositol 3-kinase/Akt in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether p70 S6 kinase is involved in TNF-␣-stimulated IL-6 synthesis in MC3T3-E1 cells. TNF-␣ time dependently induced the phosphorylation of p70 S6 kinase. Rapamycin, an inhibitor of p70 S6 kinase, which attenuated the phosphorylation of p70 S6 kinase induced by TNF-␣, significantly amplified the TNF-␣-stimulated IL-6 synthesis. TNF-␣-induced phosphorylations of both p44/p42 MAP kinase and Akt were markedly enhanced by rapamycin. The amplification by rapamycin of TNF-␣-induced IL-6 synthesis was reduced by PD98059, a specific inhibitor of MEK1/2, or Akt inhibitor. Rapamycin enhanced the IL-6 synthesis and the phosphorylation of Akt induced by TNF-␣ also in human osteoblasts. Taken together, these results strongly suggest that p70 S6 kinase limits the TNF-␣-stimulated IL-6 synthesis at a point upstream from p44/p42 MAP kinase and Akt in osteoblast-like cells. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Interleukin-6 (IL-6), which is a multifunctional cytokine inducing important physiological effects on a wide range of functions such as promoting B cell differentiation, T cell activation and inducing acute phase proteins (Akira et al., 1993; Heymann and Rousselle, 2000; Kwan Tat et al., 2004), is regarded as one of the most potent osteoclastogenic factor (Ishimi et al., 1990; Roodman, 1992; Akira et al., 1993; Kwan Tat et al., 2004). Bone metabolism is strictly regulated by two types of functional cells, osteoblasts and osteoclasts, responsible for bone formation and bone resorption, respectively (Nijweide et al., 1986). It is well known that receptor activator nuclear factor-B ligand (RANKL) expressed by bone resorptive agents on osteoblasts plays a pivotal role to transduce an essential differentiation signal to osteoclast lineage cells through binding to its receptor, RANK, expressed on the latter cells, suggesting that osteoblasts also regulate bone resorption (Nakashima et al., 2000). Thus, the formation of bone structures and bone remodeling results from the coupling process, bone resorption by activated osteo-
∗ Corresponding author. Tel.: +81 58 230 6214; fax: +81 58 230 6215. E-mail address: [email protected] (O. Kozawa). 0303-7207/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2009.10.005
clasts with subsequent deposition of new matrix by osteoblasts. Bone resorption is promoted by the increased local production of inflammatory cytokines such as TNF-␣ and IL-1. In osteoblasts, it has been reported that bone resorptive agents including TNF-␣ and IL-1 stimulate the synthesis of IL-6 (Helle et al., 1988; Ishimi et al., 1990; Littlewood et al., 1991). As for IL-6, it has been reported that IL-6 deficient female mice are protected from bone loss caused by estrogen depletion (Poli et al., 1994). We have previously shown that TNF-␣ stimulates IL-6 synthesis via phosphatidylinositol 3kinase/Akt in addition to p44/p42 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells (Takai et al., 2006). Therefore, it is now recognized that IL-6 secreted from osteoblasts takes part in bone metabolism as a downstream effector of bone resorptive agents. It is well known that p70 S6 kinase is a mitogen-activated serine/threonine kinase, which is required for cell proliferation and G1 cell cycle progression (Pullen and Thomas, 1997). In osteoblasts, it has been shown that fluoroaluminate up-regulates p70 S6 kinase phosphorylation (Susa et al., 1997). In a previous study (Kozawa et al., 2001), we showed that p70 S6 kinase takes a part in bone morphogenetic protein-4-stimulated vascular endothelial growth factor synthesis as a positive regulator in osteoblast-like MC3T3E1 cells. In addition, we have recently reported that p70 S6 kinase
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plays a role as a negative regulator in platelet-derived growth factor BB-stimulated synthesis of IL-6 in MC3T3-E1 cells (Takai et al., 2007). Regarding about p70 S6 kinase in TNF-␣ signaling, it has been shown that p70 S6 kinase activated by TNF-␣ is involved in the negative feedback loop of insulin signaling in adipocytes and hepatocytes (Zhang et al., 2008). Moreover, TNF-␣ reportedly activates Akt and its downstream effectors GSK-3, p70 S6 kinase, resulting in the increase of protein synthesis and viability in skeletal myotubes (Plaisance et al., 2008). However, the exact role of p70 S6 kinase in osteoblasts has not yet been fully elucidated. In this study, we investigated the involvement of p70 S6 kinase in the TNF-␣-stimulated IL-6 synthesis in osteoblast-like MC3T3-E1 cells. We show here that p70 S6 kinase activated by TNF-␣ negatively regulates IL-6 synthesis at a point upstream from p44/p42 MAP kinase and Akt in these cells. 2. Materials and methods 2.1. Materials Normal Human osteoblasts (NHOst) were purchased from CAMBREX (Charles, IA). Mouse and human IL-6 enzyme-linked immunosorbent assay (ELISA) kits were purchased from R&D Systems, Inc. (Minneapolis, MN). TNF-␣ was obtained from Funakoshi Pharmaceutical Co. (Tokyo, Japan). Rapamycin was obtained from Calbiochem-Novabiochem Co. (La Jolla, CA). Phospho-specific p70 S6 kinase antibodies, p70 S6 kinase antibodies, phospho-specific p44/p42 MAP kinase antibodies, p44/p42 MAP kinase antibodies, phospho-specific Akt antibodies and Akt antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA). ECL Western blotting detection system was purchased from GE Healthcare UK Ltd. (Buckinghamshire, UK). Other materials and chemicals were obtained from commercial sources. Rapamycin was dissolved in dimethyl sulfoxide. The maximum concentration of dimethyl sulfoxide was 0.1%, which did not affect the assay for IL-6 or Western blot analysis. 2.2. Cell culture Cloned osteoblast-like MC3T3-E1 cells derived from newborn mouse calvaria (Sudo et al., 1983), were maintained as previously described (Kozawa et al., 1997a,b). Briefly, the cells were cultured in ␣-minimum essential medium (␣-MEM) containing 10% fetal calf serum (FCS) at 37 ◦ C in a humidified atmosphere of 5% CO2 /95% air. The cells were seeded into 35-mm (5 × 104 cells/dish) or 90-mm (5 × 105 cells/dish) diameter dishes. After 5 days, the medium was exchanged for ␣-MEM containing 0.3% FCS. The cells were used for experiments after 48 h. NHOst were seeded into 35mm (5 × 104 cells/dish) or 90-mm (5 × 105 cells/dish) in ␣-MEM containing 10% FCS. The cells from second to third passage were used for all experiments. Forty-eight hours before the experiments, the medium was changed to ␣-MEM containing 0.3% FCS. 2.3. Assay for IL-6 The cultured cells were stimulated by various doses of TNF-␣ in 1 ml of ␣-MEM containing 0.3% FCS for the indicated periods. When indicated, the cells were pretreated with rapamycin for 60 min. The conditioned medium was collected at the end of the incubation, and the IL-6 concentration was measured by mouse or human IL-6 ELISA kit according to the manufacturer’s instruction. The assay kits can detect the corresponding IL-6 in the range between 7.8 and 500 pg/ml. When the samples generate values higher than 500 pg/ml, the samples were adequately diluted with calibrator diluent provided with the kit, and re-assayed. 2.4. Western blot analysis The cultured cells were stimulated by TNF-␣ in ␣-MEM containing 0.3% FCS for the indicated periods. When indicated, the cells were pretreated with rapamycin for 60 min. The cells were washed twice with phosphate-buffered saline and then lysed, homogenized and sonicated in a lysis buffer containing 62.5 mM Tris/HCl, pH 6.8, 2% sodium dodecyl sulfate (SDS), 50 mM dithiothreitol and 10% glycerol. The cytosolic fraction was collected as a supernatant after centrifugation at 125,000 × g for 10 min at 4 ◦ C. SDS-polyacrylamide gel electrophoresis (PAGE) was performed according to Laemmli (1970) in 10% polyacrylamide gel. Western blot analysis was performed as described previously (Kato et al., 1996) by using phospho-specific p70 S6 kinase antibodies, p70 S6 kinase antibodies, phospho-specific p44/p42 MAP kinase antibodies, p44/p42 MAP kinase antibodies, phospho-specific Akt antibodies and Akt antibodies with peroxidase-labeled antibodies raised in goat against rabbit IgG being used as second antibodies. Peroxidase activity on the PVDF sheet was visualized on X-ray film by means of the ECL Western blot detection system.
Fig. 1. Effect of TNF-␣ on the phosphorylation of p70 S6 kinase in MC3T3-E1 cells. The cultured cells were stimulated with 30 ng/ml TNF-␣ for the indicated periods. The extracts of cells were subjected to SDS-PAGE with subsequent Western blotting analysis with antibodies against phospho-specific p70 S6 kinase or p70 S6 kinase.
2.5. Determination The absorbance of ELISA samples was measured at 450 nm with EL 340 Bio Kinetic Reader (Bio-Tek Instruments, Inc., Winooski, VT).
2.6. Statistical analysis The data were analyzed by ANOVA followed by the Bonferroni method for multiple comparisons between pairs, and a p < 0.05 was considered significant. All data are presented as the mean ± SEM of triplicate determinations from three independent cell preparations. Each experiment was repeated three times with similar results.
3. Results 3.1. Effect of TNF-˛ on the phosphorylation of p70 S6 kinase in MC3T3-E1 cells In order to clarify whether TNF-␣ activates p70 S6 kinase in osteoblast-like MC3T3-E1 cells, we examined the effect of TNF-␣ on the phosphorylation of p70 S6 kinase, the activated form of p70 S6 kinase, using phospho-specific p70 S6 kinase (Thr389) antibodies. The phosphorylation of p70 S6 kinase was markedly observed from 5 min after the TNF-␣-stimulation, and still observed at 30 min after the stimulation (Fig. 1).
3.2. Effect of rapamycin on the TNF-˛-stimulated IL-6 synthesis in osteoblast-like cells We have previously shown that TNF-␣ stimulates synthesis of IL-6 through the activation of p44/p42 MAP kinase and PI3kinase/Akt in osteoblast-like MC3T3-E1 cells (Takai et al., 2006). In order to clarify the involvement of p70 S6 kinase in the TNF-␣induced synthesis of IL-6 in MC3T3-E1 cells or not, we examined the effect of rapamycin, a specific inhibitor of p70 S6 kinase (Price et al., 1992; Kuo et al., 1992), on the TNF-␣-stimulated synthesis of IL-6. Rapamycin, which by itself had no effect on the basal levels of IL-6, significantly amplified the IL-6 synthesis induced by TNF␣ at 30 ng/ml, the maximum dose for IL-6 in these cells (Kozawa et al., 1997a,b) (Fig. 2). The amplifying effect of rapamycin on the IL-6 synthesis was dose-dependent in the range between 0.3 and 30 ng/ml (Fig. 2). We also examined effect of rapamycin on IL-6 synthesis induced by TNF-␣ at 3 ng/ml, and confirmed that rapamycin significantly amplified the TNF-␣-stimulated IL-6 synthesis (data not shown). We next examined the effect of rapamycin in NHOst, a different osteoblastic cell type. We found that TNF-␣ significantly increased induced IL-6 synthesis also in these osteoblasts (25.1 ± 0.8-fold for 30 ng/ml TNF-␣ alone compared to the control, *p < 0.005). In addition, rapamycin significantly increased TNF-␣-stimulated IL-6 synthesis (1.1 ± 0.0-fold for 30 ng/ml TNF-␣ with 3 ng/ml rapamycin compared to the value of TNF-␣ alone, *p < 0.05).
C. Minamitani et al. / Molecular and Cellular Endocrinology 315 (2010) 195–200
Fig. 2. Effect of rapamycin on the TNF-␣-stimulated IL-6 synthesis in MC3T3-E1 cells. The cultured cells were pretreated with various doses of rapamycin for 60 min, and then stimulated by 30 ng/ml TNF-␣ (䊉) or vehicle () for 48 h. Each value represents the mean ± SEM of triplicate determinations from three independent cell preparations. Similar results were obtained with two additional and different cell preparations. *p < 0.05, compared to the value of TNF-␣ alone.
3.3. Effect of rapamycin on the phosphorylation of p70 S6 kinase in MC3T3-E1 cells We examined the effect of rapamycin on the TNF-␣-induced phosphorylation of p70 S6 kinase in MC3T3-E1 cells. Rapamycin, which alone did not affect the phosphorylation of p70 S6 kinase, truly suppressed the TNF-␣-induced phosphorylation of p70 S6 kinase (Fig. 3). 3.4. Effect of rapamycin on the TNF-˛-induced phosphorylation of p44/p42 MAP kinase and Akt in osteoblast-like cells To investigate whether the amplification of the TNF-␣stimulated IL-6 synthesis by rapamycin is dependent on the activation of p44/p42 MAP kinase or Akt in MC3T3-E1 cells, we next examined the effect of rapamycin on the TNF-␣-induced phosphorylation of p44/p42 MAP kinase and Akt, the activated forms of
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Fig. 4. Effect of rapamycin on the TNF-␣-induced phosphorylation of p44/p42 MAP kinase in MC3T3-E1 cells. The cultured cells were pretreated with various doses of rapamycin for 60 min, and then stimulated by 30 ng/ml TNF-␣ or vehicle for 10 min. The extracts of cells were subjected to SDS-PAGE with subsequent Western blotting analysis with antibodies against phospho-specific p44/p42 MAP kinase or p44/p42 MAP kinase. The histogram shows the quantitative representations of the levels of TNF-␣-induced phosphorylation obtained from laser densitometric analysis of three independent experiments. Each value represents the mean ± SEM of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *p < 0.05, compared to the control. **p < 0.05, compared to the value of TNF-␣ alone.
these kinases. Rapamycin markedly enhanced the TNF-␣-induced phosphorylation of p44/p42 MAP kinase (Fig. 4) and Akt (Fig. 5). We next examined the effect of rapamycin on the TNF-␣-induced phosphorylation of p44/p42 MAP kinase and Akt in NHOst. TNF-␣ itself induced the phosphorylation of Akt and rapamycin markedly enhanced it in these cells (Fig. 6). In contrast, rapamycin failed to affect TNF-␣-induced phosphorylation of p44/p42 MAP kinase (data not shown). 3.5. Effect of PD98059 on the amplification by rapamycin of the TNF-˛-stimulated IL-6 synthesis in MC3T3-E1 cells We next examined the effect of PD98059, a specific inhibitor of MEK1/2, upstream kinase that activates p44/p42 MAP kinase (Alessi et al., 1995), on the enhancement by rapamycin of TNF␣-induced IL-6 synthesis in MC3T3-E1 cells. PD98059, which by itself did not affect the basal levels of IL-6, significantly reduced the enhancement by rapamycin of IL-6 synthesis induced by TNF␣ (Table 1). PD98059 (50 M) caused about 70% reduction in the rapamycin-effect. 3.6. Effect of Akt inhibitor on the amplification by rapamycin of the TNF-˛-stimulated IL-6 synthesis in MC3T3-E1 cells
Fig. 3. Effect of rapamycin on the TNF-␣-induced phosphorylation of p70 S6 kinase in MC3T3-E1 cells. The cultured cells were pretreated with 30 ng/ml rapamycin or vehicle for 60 min, and then stimulated by 30 ng/ml TNF-␣ or vehicle for 10 min. The extracts of cells were subjected to SDS-PAGE with subsequent Western blotting analysis with antibodies against phospho-specific p70 S6 kinase or p70 S6 kinase.
In addition, we examined the effect of Akt inhibitor, 1L-6-hydroxymethyl-chiro-inositol-2-(R)-2-O-methyl-3-Ooctadecylcarbonate (Hu et al., 2000), on the rapamycin-effect
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Fig. 5. Effect of rapamycin on the TNF-␣-induced phosphorylation of Akt in MC3T3E1 cells. The cultured cells were pretreated with various doses of rapamycin for 60 min, and then stimulated by 30 ng/ml TNF-␣ or vehicle for 10 min. The extracts of cells were subjected to SDS-PAGE with subsequent Western blotting analysis with antibodies against phospho-specific Akt or Akt. The histogram shows quantitative representations of the levels of TNF-␣-induced phosphorylation obtained from laser densitometric analysis of three independent experiments. Each value represents the mean ± SEM of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *p < 0.05, compared to the control. **p < 0.05, compared to the value of TNF-␣ alone.
in MC3T3-E1 cells. Akt inhibitor, which alone did not affect the basal levels of IL-6, suppressed the enhancement by rapamycin of TNF-␣-induced IL-6 synthesis (Table 2). Akt inhibitor (30 M) caused about 70% reduction of the enhancement. 4. Discussion In the present study, we showed that TNF-␣ induced the phosphorylation of p70 S6 kinase in osteoblast-like MC3T3-E1 cells, using phospho-specific p70 S6 kinase (Thr389) antibodies. It has been reported that phosphorylation at Thr389 most strongly correlates with the activation of p70 S6 kinase among the phosphorylation sites (Pullen and Thomas, 1997). Therefore, our findings suggest that TNF-␣ stimulates the activation of p70 S6 kinase in osteoblast-like MC3T3-E1 cells. It is well known that the p70 S6 kinase pathway plays an important role in various cellular functions, especially cell cycle progression (Pullen and Thomas, 1997). In osteoblast-like MC3T3-E1 cells, we have previously reported that p70 S6 kinase negatively regulates platelet-derived growth factor BB-stimulated IL-6 synthesis, a key factor in bone remodeling (Takai et al., 2007). In addition, we have reported that TNF-␣ stimulates IL-6 synthesis via phosphatidylinositol 3-kinase/Akt in addition to p44/p42 MAP kinase in these cells (Takai et al., 2006). Therefore, in the present study, we investigated the involvement of p70 S6 kinase in the TNF-␣-stimulated IL-6 synthesis or not in osteoblast-like MC3T3-E1 cells. We found that rapamycin (Price et al., 1992; Kuo et al., 1992) significantly amplified the IL-6 syn-
Fig. 6. Effect of rapamycin on the TNF-␣-induced phosphorylation of Akt in NHOst. The cultured cells were pretreated with 50 ng/ml rapamycin for 60 min, and then stimulated by 30 ng/ml TNF-␣ or vehicle for 10 min. The extracts of cells were subjected to SDS-PAGE with subsequent Western blotting analysis with antibodies against phospho-specific Akt or Akt. The histogram shows quantitative representations of the levels of TNF-␣-induced phosphorylation obtained from laser densitometric analysis of three independent experiments. Each value represents the mean ± SEM of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *p < 0.05, compared to the control. **p < 0.05, compared to the value of TNF-␣ alone.
thesis induced by TNF-␣ in these cells. We confirmed here that rapamycin truly suppressed TNF-␣-activated p70 S6 kinase. These results suggest that the suppression of p70 S6 kinase by rapamycin causes up-regulation of TNF-␣-stimulated IL-6 synthesis. Based on our findings, it is most likely that TNF-␣-induced IL-6 synthesis is negatively regulated by p70 S6 kinase which is activated by TNF-␣ itself in osteoblast-like MC3T3-E1 cells (Fig. 7). It is well recognized that Akt mediates intracellular signaling of extracellular agonists and plays a crucial role in cellular funcTable 1 Effect of a combination of PD98059 and rapamycin on the TNF-␣-stimulated IL-6 synthesis in MC3T3-E1 cells. PD98059
Rapamycin
TNF-␣
IL-6 (pg/ml)
− − − − + + + +
− − + + − − + +
− + − + − + − +
10 439 39 2361 4 279 33 743
± ± ± ± ± ± ± ±
1 34* 3 40** 0 21** 0 9***
The cultured cells were pretreated with 50 M PD98059, 30 ng/ml rapamycin or vehicle for 60 min, and then stimulated by 30 ng/ml TNF-␣ for 48 h. Each value represents the mean ± SEM of triplicate determinations. Similar results were obtained with two additional and different cell preparations. * p < 0.05 compared to the control. ** p < 0.05 compared to the value of TNF-␣ alone. *** p < 0.05 compared to the value of TNF-␣.
C. Minamitani et al. / Molecular and Cellular Endocrinology 315 (2010) 195–200 Table 2 Effect of a combination of Akt inhibitor and rapamycin on the TNF-␣-stimulated IL-6 synthesis in MC3T3-E1 cells. Akt inhibitor
Rapamycin
TNF-␣
IL-6 (pg/ml)
− − − − + + + +
− − + + − − + +
− + − + − + − +
7 414 6 1864 4 203 13 646
± ± ± ± ± ± ± ±
2 29* 1 98** 1 24** 3 40***
The cultured cells were pretreated with 30 M Akt inhibitor, 30 ng/ml rapamycin or vehicle for 60 min, and then stimulated by 30 ng/ml TNF-␣ for 48 h. Each value represents the mean ± SEM of triplicate determinations. Similar results were obtained with two additional and different cell preparations. * p < 0.05 compared to the control. ** p < 0.05 compared to the value of TNF-alone. *** p < 0.05 compared to the value of TNF-␣ with rapamycin.
tions such as proliferation and cell survival in a variety of cells (Coffer et al., 1998). Akt is activated by the phosphorylation of serine residues (Coffer et al., 1998). In this study, rapamycin enhanced the TNF-␣-induced phosphorylation of both p44/p42 MAP kinase and Akt in osteoblast-like MC3T3-E1 cells. In addition, we found that Akt inhibitor (Hu et al., 2000) not only suppressed the TNF␣-stimulated IL-6 synthesis but also reduced the amplification by rapamycin of the TNF-␣-stimulated IL-6 synthesis. Moreover, PD98059 (Alessi et al., 1995) as well as Akt inhibitor, reduced the rapamycin-induced enhancement in TNF-␣-stimulated synthesis of IL-6. Taken together, these findings provides compelling evidence that p70 S6 kinase negatively regulates the TNF-␣stimulated IL-6 synthesis at a point upstream of p44/p42 MAP kinase and Akt in osteoblast-like MC3T3-E1 cells. We also found that rapamycin enhanced the IL-6 synthesis in addition to the phosphorylation of Akt induced by TNF-␣ in NHOst, a different osteoblastic cell type. Therefore, it is likely that negative regulation by p70 S6K of the TNF-␣-stimulated IL-6 synthesis through Akt is common in osteoblasts. The potential mechanism of regulation by p70 S6 kinase in TNF-␣-stimulated IL-6 synthesis in osteoblasts shown here is summarized in Fig. 6. It has been shown that TNF-␣ increases the expression of IL-6 gene at least in part
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through activation of nuclear factor-B (NF-B) in osteoblast-like cells (Kurokouchi et al., 1998). In addition, tumor necrosis factor receptor (TNFR) 2 reportedly facilitates phosphatidylinositol 3-kinase-dependent NF-B activation (Marchetti et al., 2004). It might be possible that NF-B activation is also regulated by p70 S6 kinase in osteoblasts. Further investigation would be required to clarify the details. It is generally known that the inflammatory cytokines such as IL-1, IL-6 and TNF-␣ secreted from osteoblasts play pivotal roles in the pathogenesis of postmenopausal osteoporosis through upregulating osteoclasts recruitment and their activities (Manolagas and Jilka, 1995). In addition, over-expression of inflammatory cytokines is well known to be involved in the important clinical feature of inflammatory arthritis including rheumatoid arthritis (Deleuren et al., 1992; Gravallese et al., 2000; Lam et al., 2000; Shigeyama et al., 2000; Wei et al., 2005). In the last decade, monoclonal antibody against TNF-␣ such as infliximab has shown clinical efficacy in controlling the inflammatory signs and symptoms of rheumatoid arthritis, indicating the importance of TNF-␣-signaling in the pathogenesis. Our present findings, indicating that the p70 S6 kinase signaling activated by TNF-␣ limits the TNF-␣-induced oversynthesis of IL-6 in osteoblast-like MC3T3-E1 cells, might provide a new aspect of therapeutics for both metabolic and inflammatory bone diseases. The adequate regulation of the p70 S6 kinase pathway in osteoblasts, as well as the TNF-␣ pathway, could be a new strategy of pathological bone resorption concurrent with these diseases. In conclusion, our results strongly suggest that p70 S6 kinase negatively regulates TNF-␣-induced IL-6 synthesis at a point upstream of p44/p42 MAP kinase and Akt in osteoblast cell lines. Further research to confirm this in osteoblasts themselves is needed. Acknowledgements We are very grateful to Yoko Kawamura for her skillful technical assistance. This investigation was supported in part by Grant-in-Aid for Scientific Research (16590873 and 16591482) for the Ministry of Education, Science, Sports and Culture of Japan, the Foundation for Growth Science, Health and Labour Sciences, the Research Grants for Research on Proteomics and Research on Longevity Sciences from the Ministry of Health, Labour and Welfare of Japan. References
Fig. 7. Schematic representation of the involvement of p70 S6 kinase in the mechanism of the TNF-␣-stimulated IL-6 synthesis in osteoblast-like MC3T3-E1 cells. Abbreviations used are as follows: TNF-␣, tumor necrosis factor-␣; MAP kinase, mitogen-activated protein; IL-6, interleukin-6.
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Molecular and Cellular Endocrinology journal homepage: www.elsevier.com/locate/mce
p70 S6 kinase limits tumor necrosis factor-␣-induced interleukin-6 synthesis in osteoblast-like cells Chiho Minamitani a,b , Haruhiko Tokuda b,c , Seiji Adachi b , Rie Matsushima-Nishiwaki b , Junichi Yamauchi b , Kenji Kato a,b , Hideo Natsume a,b , Jun Mizutani a , Osamu Kozawa b,∗ , Takanobu Otsuka a a
Department of Orthopedic Surgery, Nagoya City University Graduate School of Medical Sciences, Nagoya 467-8601, Japan Department of Pharmacology, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu 501-1194, Japan c Department of Clinical Laboratory, National Hospital for Geriatric Medicine, National Center for Geriatrics and Gerontology, Obu 474-8511, Japan b
a r t i c l e
i n f o
Article history: Received 11 June 2009 Received in revised form 7 October 2009 Accepted 16 October 2009 Keywords: TNF-␣ p70 S6 kinase IL-6 Osteoblast
a b s t r a c t Our previous study demonstrated that tumor necrosis factor-␣ (TNF-␣) stimulates the synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, via p44/p42 mitogen-activated protein (MAP) kinase and phosphatidylinositol 3-kinase/Akt in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether p70 S6 kinase is involved in TNF-␣-stimulated IL-6 synthesis in MC3T3-E1 cells. TNF-␣ time dependently induced the phosphorylation of p70 S6 kinase. Rapamycin, an inhibitor of p70 S6 kinase, which attenuated the phosphorylation of p70 S6 kinase induced by TNF-␣, significantly amplified the TNF-␣-stimulated IL-6 synthesis. TNF-␣-induced phosphorylations of both p44/p42 MAP kinase and Akt were markedly enhanced by rapamycin. The amplification by rapamycin of TNF-␣-induced IL-6 synthesis was reduced by PD98059, a specific inhibitor of MEK1/2, or Akt inhibitor. Rapamycin enhanced the IL-6 synthesis and the phosphorylation of Akt induced by TNF-␣ also in human osteoblasts. Taken together, these results strongly suggest that p70 S6 kinase limits the TNF-␣-stimulated IL-6 synthesis at a point upstream from p44/p42 MAP kinase and Akt in osteoblast-like cells. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Interleukin-6 (IL-6), which is a multifunctional cytokine inducing important physiological effects on a wide range of functions such as promoting B cell differentiation, T cell activation and inducing acute phase proteins (Akira et al., 1993; Heymann and Rousselle, 2000; Kwan Tat et al., 2004), is regarded as one of the most potent osteoclastogenic factor (Ishimi et al., 1990; Roodman, 1992; Akira et al., 1993; Kwan Tat et al., 2004). Bone metabolism is strictly regulated by two types of functional cells, osteoblasts and osteoclasts, responsible for bone formation and bone resorption, respectively (Nijweide et al., 1986). It is well known that receptor activator nuclear factor-B ligand (RANKL) expressed by bone resorptive agents on osteoblasts plays a pivotal role to transduce an essential differentiation signal to osteoclast lineage cells through binding to its receptor, RANK, expressed on the latter cells, suggesting that osteoblasts also regulate bone resorption (Nakashima et al., 2000). Thus, the formation of bone structures and bone remodeling results from the coupling process, bone resorption by activated osteo-
∗ Corresponding author. Tel.: +81 58 230 6214; fax: +81 58 230 6215. E-mail address: [email protected] (O. Kozawa). 0303-7207/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2009.10.005
clasts with subsequent deposition of new matrix by osteoblasts. Bone resorption is promoted by the increased local production of inflammatory cytokines such as TNF-␣ and IL-1. In osteoblasts, it has been reported that bone resorptive agents including TNF-␣ and IL-1 stimulate the synthesis of IL-6 (Helle et al., 1988; Ishimi et al., 1990; Littlewood et al., 1991). As for IL-6, it has been reported that IL-6 deficient female mice are protected from bone loss caused by estrogen depletion (Poli et al., 1994). We have previously shown that TNF-␣ stimulates IL-6 synthesis via phosphatidylinositol 3kinase/Akt in addition to p44/p42 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells (Takai et al., 2006). Therefore, it is now recognized that IL-6 secreted from osteoblasts takes part in bone metabolism as a downstream effector of bone resorptive agents. It is well known that p70 S6 kinase is a mitogen-activated serine/threonine kinase, which is required for cell proliferation and G1 cell cycle progression (Pullen and Thomas, 1997). In osteoblasts, it has been shown that fluoroaluminate up-regulates p70 S6 kinase phosphorylation (Susa et al., 1997). In a previous study (Kozawa et al., 2001), we showed that p70 S6 kinase takes a part in bone morphogenetic protein-4-stimulated vascular endothelial growth factor synthesis as a positive regulator in osteoblast-like MC3T3E1 cells. In addition, we have recently reported that p70 S6 kinase
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plays a role as a negative regulator in platelet-derived growth factor BB-stimulated synthesis of IL-6 in MC3T3-E1 cells (Takai et al., 2007). Regarding about p70 S6 kinase in TNF-␣ signaling, it has been shown that p70 S6 kinase activated by TNF-␣ is involved in the negative feedback loop of insulin signaling in adipocytes and hepatocytes (Zhang et al., 2008). Moreover, TNF-␣ reportedly activates Akt and its downstream effectors GSK-3, p70 S6 kinase, resulting in the increase of protein synthesis and viability in skeletal myotubes (Plaisance et al., 2008). However, the exact role of p70 S6 kinase in osteoblasts has not yet been fully elucidated. In this study, we investigated the involvement of p70 S6 kinase in the TNF-␣-stimulated IL-6 synthesis in osteoblast-like MC3T3-E1 cells. We show here that p70 S6 kinase activated by TNF-␣ negatively regulates IL-6 synthesis at a point upstream from p44/p42 MAP kinase and Akt in these cells. 2. Materials and methods 2.1. Materials Normal Human osteoblasts (NHOst) were purchased from CAMBREX (Charles, IA). Mouse and human IL-6 enzyme-linked immunosorbent assay (ELISA) kits were purchased from R&D Systems, Inc. (Minneapolis, MN). TNF-␣ was obtained from Funakoshi Pharmaceutical Co. (Tokyo, Japan). Rapamycin was obtained from Calbiochem-Novabiochem Co. (La Jolla, CA). Phospho-specific p70 S6 kinase antibodies, p70 S6 kinase antibodies, phospho-specific p44/p42 MAP kinase antibodies, p44/p42 MAP kinase antibodies, phospho-specific Akt antibodies and Akt antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA). ECL Western blotting detection system was purchased from GE Healthcare UK Ltd. (Buckinghamshire, UK). Other materials and chemicals were obtained from commercial sources. Rapamycin was dissolved in dimethyl sulfoxide. The maximum concentration of dimethyl sulfoxide was 0.1%, which did not affect the assay for IL-6 or Western blot analysis. 2.2. Cell culture Cloned osteoblast-like MC3T3-E1 cells derived from newborn mouse calvaria (Sudo et al., 1983), were maintained as previously described (Kozawa et al., 1997a,b). Briefly, the cells were cultured in ␣-minimum essential medium (␣-MEM) containing 10% fetal calf serum (FCS) at 37 ◦ C in a humidified atmosphere of 5% CO2 /95% air. The cells were seeded into 35-mm (5 × 104 cells/dish) or 90-mm (5 × 105 cells/dish) diameter dishes. After 5 days, the medium was exchanged for ␣-MEM containing 0.3% FCS. The cells were used for experiments after 48 h. NHOst were seeded into 35mm (5 × 104 cells/dish) or 90-mm (5 × 105 cells/dish) in ␣-MEM containing 10% FCS. The cells from second to third passage were used for all experiments. Forty-eight hours before the experiments, the medium was changed to ␣-MEM containing 0.3% FCS. 2.3. Assay for IL-6 The cultured cells were stimulated by various doses of TNF-␣ in 1 ml of ␣-MEM containing 0.3% FCS for the indicated periods. When indicated, the cells were pretreated with rapamycin for 60 min. The conditioned medium was collected at the end of the incubation, and the IL-6 concentration was measured by mouse or human IL-6 ELISA kit according to the manufacturer’s instruction. The assay kits can detect the corresponding IL-6 in the range between 7.8 and 500 pg/ml. When the samples generate values higher than 500 pg/ml, the samples were adequately diluted with calibrator diluent provided with the kit, and re-assayed. 2.4. Western blot analysis The cultured cells were stimulated by TNF-␣ in ␣-MEM containing 0.3% FCS for the indicated periods. When indicated, the cells were pretreated with rapamycin for 60 min. The cells were washed twice with phosphate-buffered saline and then lysed, homogenized and sonicated in a lysis buffer containing 62.5 mM Tris/HCl, pH 6.8, 2% sodium dodecyl sulfate (SDS), 50 mM dithiothreitol and 10% glycerol. The cytosolic fraction was collected as a supernatant after centrifugation at 125,000 × g for 10 min at 4 ◦ C. SDS-polyacrylamide gel electrophoresis (PAGE) was performed according to Laemmli (1970) in 10% polyacrylamide gel. Western blot analysis was performed as described previously (Kato et al., 1996) by using phospho-specific p70 S6 kinase antibodies, p70 S6 kinase antibodies, phospho-specific p44/p42 MAP kinase antibodies, p44/p42 MAP kinase antibodies, phospho-specific Akt antibodies and Akt antibodies with peroxidase-labeled antibodies raised in goat against rabbit IgG being used as second antibodies. Peroxidase activity on the PVDF sheet was visualized on X-ray film by means of the ECL Western blot detection system.
Fig. 1. Effect of TNF-␣ on the phosphorylation of p70 S6 kinase in MC3T3-E1 cells. The cultured cells were stimulated with 30 ng/ml TNF-␣ for the indicated periods. The extracts of cells were subjected to SDS-PAGE with subsequent Western blotting analysis with antibodies against phospho-specific p70 S6 kinase or p70 S6 kinase.
2.5. Determination The absorbance of ELISA samples was measured at 450 nm with EL 340 Bio Kinetic Reader (Bio-Tek Instruments, Inc., Winooski, VT).
2.6. Statistical analysis The data were analyzed by ANOVA followed by the Bonferroni method for multiple comparisons between pairs, and a p < 0.05 was considered significant. All data are presented as the mean ± SEM of triplicate determinations from three independent cell preparations. Each experiment was repeated three times with similar results.
3. Results 3.1. Effect of TNF-˛ on the phosphorylation of p70 S6 kinase in MC3T3-E1 cells In order to clarify whether TNF-␣ activates p70 S6 kinase in osteoblast-like MC3T3-E1 cells, we examined the effect of TNF-␣ on the phosphorylation of p70 S6 kinase, the activated form of p70 S6 kinase, using phospho-specific p70 S6 kinase (Thr389) antibodies. The phosphorylation of p70 S6 kinase was markedly observed from 5 min after the TNF-␣-stimulation, and still observed at 30 min after the stimulation (Fig. 1).
3.2. Effect of rapamycin on the TNF-˛-stimulated IL-6 synthesis in osteoblast-like cells We have previously shown that TNF-␣ stimulates synthesis of IL-6 through the activation of p44/p42 MAP kinase and PI3kinase/Akt in osteoblast-like MC3T3-E1 cells (Takai et al., 2006). In order to clarify the involvement of p70 S6 kinase in the TNF-␣induced synthesis of IL-6 in MC3T3-E1 cells or not, we examined the effect of rapamycin, a specific inhibitor of p70 S6 kinase (Price et al., 1992; Kuo et al., 1992), on the TNF-␣-stimulated synthesis of IL-6. Rapamycin, which by itself had no effect on the basal levels of IL-6, significantly amplified the IL-6 synthesis induced by TNF␣ at 30 ng/ml, the maximum dose for IL-6 in these cells (Kozawa et al., 1997a,b) (Fig. 2). The amplifying effect of rapamycin on the IL-6 synthesis was dose-dependent in the range between 0.3 and 30 ng/ml (Fig. 2). We also examined effect of rapamycin on IL-6 synthesis induced by TNF-␣ at 3 ng/ml, and confirmed that rapamycin significantly amplified the TNF-␣-stimulated IL-6 synthesis (data not shown). We next examined the effect of rapamycin in NHOst, a different osteoblastic cell type. We found that TNF-␣ significantly increased induced IL-6 synthesis also in these osteoblasts (25.1 ± 0.8-fold for 30 ng/ml TNF-␣ alone compared to the control, *p < 0.005). In addition, rapamycin significantly increased TNF-␣-stimulated IL-6 synthesis (1.1 ± 0.0-fold for 30 ng/ml TNF-␣ with 3 ng/ml rapamycin compared to the value of TNF-␣ alone, *p < 0.05).
C. Minamitani et al. / Molecular and Cellular Endocrinology 315 (2010) 195–200
Fig. 2. Effect of rapamycin on the TNF-␣-stimulated IL-6 synthesis in MC3T3-E1 cells. The cultured cells were pretreated with various doses of rapamycin for 60 min, and then stimulated by 30 ng/ml TNF-␣ (䊉) or vehicle () for 48 h. Each value represents the mean ± SEM of triplicate determinations from three independent cell preparations. Similar results were obtained with two additional and different cell preparations. *p < 0.05, compared to the value of TNF-␣ alone.
3.3. Effect of rapamycin on the phosphorylation of p70 S6 kinase in MC3T3-E1 cells We examined the effect of rapamycin on the TNF-␣-induced phosphorylation of p70 S6 kinase in MC3T3-E1 cells. Rapamycin, which alone did not affect the phosphorylation of p70 S6 kinase, truly suppressed the TNF-␣-induced phosphorylation of p70 S6 kinase (Fig. 3). 3.4. Effect of rapamycin on the TNF-˛-induced phosphorylation of p44/p42 MAP kinase and Akt in osteoblast-like cells To investigate whether the amplification of the TNF-␣stimulated IL-6 synthesis by rapamycin is dependent on the activation of p44/p42 MAP kinase or Akt in MC3T3-E1 cells, we next examined the effect of rapamycin on the TNF-␣-induced phosphorylation of p44/p42 MAP kinase and Akt, the activated forms of
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Fig. 4. Effect of rapamycin on the TNF-␣-induced phosphorylation of p44/p42 MAP kinase in MC3T3-E1 cells. The cultured cells were pretreated with various doses of rapamycin for 60 min, and then stimulated by 30 ng/ml TNF-␣ or vehicle for 10 min. The extracts of cells were subjected to SDS-PAGE with subsequent Western blotting analysis with antibodies against phospho-specific p44/p42 MAP kinase or p44/p42 MAP kinase. The histogram shows the quantitative representations of the levels of TNF-␣-induced phosphorylation obtained from laser densitometric analysis of three independent experiments. Each value represents the mean ± SEM of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *p < 0.05, compared to the control. **p < 0.05, compared to the value of TNF-␣ alone.
these kinases. Rapamycin markedly enhanced the TNF-␣-induced phosphorylation of p44/p42 MAP kinase (Fig. 4) and Akt (Fig. 5). We next examined the effect of rapamycin on the TNF-␣-induced phosphorylation of p44/p42 MAP kinase and Akt in NHOst. TNF-␣ itself induced the phosphorylation of Akt and rapamycin markedly enhanced it in these cells (Fig. 6). In contrast, rapamycin failed to affect TNF-␣-induced phosphorylation of p44/p42 MAP kinase (data not shown). 3.5. Effect of PD98059 on the amplification by rapamycin of the TNF-˛-stimulated IL-6 synthesis in MC3T3-E1 cells We next examined the effect of PD98059, a specific inhibitor of MEK1/2, upstream kinase that activates p44/p42 MAP kinase (Alessi et al., 1995), on the enhancement by rapamycin of TNF␣-induced IL-6 synthesis in MC3T3-E1 cells. PD98059, which by itself did not affect the basal levels of IL-6, significantly reduced the enhancement by rapamycin of IL-6 synthesis induced by TNF␣ (Table 1). PD98059 (50 M) caused about 70% reduction in the rapamycin-effect. 3.6. Effect of Akt inhibitor on the amplification by rapamycin of the TNF-˛-stimulated IL-6 synthesis in MC3T3-E1 cells
Fig. 3. Effect of rapamycin on the TNF-␣-induced phosphorylation of p70 S6 kinase in MC3T3-E1 cells. The cultured cells were pretreated with 30 ng/ml rapamycin or vehicle for 60 min, and then stimulated by 30 ng/ml TNF-␣ or vehicle for 10 min. The extracts of cells were subjected to SDS-PAGE with subsequent Western blotting analysis with antibodies against phospho-specific p70 S6 kinase or p70 S6 kinase.
In addition, we examined the effect of Akt inhibitor, 1L-6-hydroxymethyl-chiro-inositol-2-(R)-2-O-methyl-3-Ooctadecylcarbonate (Hu et al., 2000), on the rapamycin-effect
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Fig. 5. Effect of rapamycin on the TNF-␣-induced phosphorylation of Akt in MC3T3E1 cells. The cultured cells were pretreated with various doses of rapamycin for 60 min, and then stimulated by 30 ng/ml TNF-␣ or vehicle for 10 min. The extracts of cells were subjected to SDS-PAGE with subsequent Western blotting analysis with antibodies against phospho-specific Akt or Akt. The histogram shows quantitative representations of the levels of TNF-␣-induced phosphorylation obtained from laser densitometric analysis of three independent experiments. Each value represents the mean ± SEM of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *p < 0.05, compared to the control. **p < 0.05, compared to the value of TNF-␣ alone.
in MC3T3-E1 cells. Akt inhibitor, which alone did not affect the basal levels of IL-6, suppressed the enhancement by rapamycin of TNF-␣-induced IL-6 synthesis (Table 2). Akt inhibitor (30 M) caused about 70% reduction of the enhancement. 4. Discussion In the present study, we showed that TNF-␣ induced the phosphorylation of p70 S6 kinase in osteoblast-like MC3T3-E1 cells, using phospho-specific p70 S6 kinase (Thr389) antibodies. It has been reported that phosphorylation at Thr389 most strongly correlates with the activation of p70 S6 kinase among the phosphorylation sites (Pullen and Thomas, 1997). Therefore, our findings suggest that TNF-␣ stimulates the activation of p70 S6 kinase in osteoblast-like MC3T3-E1 cells. It is well known that the p70 S6 kinase pathway plays an important role in various cellular functions, especially cell cycle progression (Pullen and Thomas, 1997). In osteoblast-like MC3T3-E1 cells, we have previously reported that p70 S6 kinase negatively regulates platelet-derived growth factor BB-stimulated IL-6 synthesis, a key factor in bone remodeling (Takai et al., 2007). In addition, we have reported that TNF-␣ stimulates IL-6 synthesis via phosphatidylinositol 3-kinase/Akt in addition to p44/p42 MAP kinase in these cells (Takai et al., 2006). Therefore, in the present study, we investigated the involvement of p70 S6 kinase in the TNF-␣-stimulated IL-6 synthesis or not in osteoblast-like MC3T3-E1 cells. We found that rapamycin (Price et al., 1992; Kuo et al., 1992) significantly amplified the IL-6 syn-
Fig. 6. Effect of rapamycin on the TNF-␣-induced phosphorylation of Akt in NHOst. The cultured cells were pretreated with 50 ng/ml rapamycin for 60 min, and then stimulated by 30 ng/ml TNF-␣ or vehicle for 10 min. The extracts of cells were subjected to SDS-PAGE with subsequent Western blotting analysis with antibodies against phospho-specific Akt or Akt. The histogram shows quantitative representations of the levels of TNF-␣-induced phosphorylation obtained from laser densitometric analysis of three independent experiments. Each value represents the mean ± SEM of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *p < 0.05, compared to the control. **p < 0.05, compared to the value of TNF-␣ alone.
thesis induced by TNF-␣ in these cells. We confirmed here that rapamycin truly suppressed TNF-␣-activated p70 S6 kinase. These results suggest that the suppression of p70 S6 kinase by rapamycin causes up-regulation of TNF-␣-stimulated IL-6 synthesis. Based on our findings, it is most likely that TNF-␣-induced IL-6 synthesis is negatively regulated by p70 S6 kinase which is activated by TNF-␣ itself in osteoblast-like MC3T3-E1 cells (Fig. 7). It is well recognized that Akt mediates intracellular signaling of extracellular agonists and plays a crucial role in cellular funcTable 1 Effect of a combination of PD98059 and rapamycin on the TNF-␣-stimulated IL-6 synthesis in MC3T3-E1 cells. PD98059
Rapamycin
TNF-␣
IL-6 (pg/ml)
− − − − + + + +
− − + + − − + +
− + − + − + − +
10 439 39 2361 4 279 33 743
± ± ± ± ± ± ± ±
1 34* 3 40** 0 21** 0 9***
The cultured cells were pretreated with 50 M PD98059, 30 ng/ml rapamycin or vehicle for 60 min, and then stimulated by 30 ng/ml TNF-␣ for 48 h. Each value represents the mean ± SEM of triplicate determinations. Similar results were obtained with two additional and different cell preparations. * p < 0.05 compared to the control. ** p < 0.05 compared to the value of TNF-␣ alone. *** p < 0.05 compared to the value of TNF-␣.
C. Minamitani et al. / Molecular and Cellular Endocrinology 315 (2010) 195–200 Table 2 Effect of a combination of Akt inhibitor and rapamycin on the TNF-␣-stimulated IL-6 synthesis in MC3T3-E1 cells. Akt inhibitor
Rapamycin
TNF-␣
IL-6 (pg/ml)
− − − − + + + +
− − + + − − + +
− + − + − + − +
7 414 6 1864 4 203 13 646
± ± ± ± ± ± ± ±
2 29* 1 98** 1 24** 3 40***
The cultured cells were pretreated with 30 M Akt inhibitor, 30 ng/ml rapamycin or vehicle for 60 min, and then stimulated by 30 ng/ml TNF-␣ for 48 h. Each value represents the mean ± SEM of triplicate determinations. Similar results were obtained with two additional and different cell preparations. * p < 0.05 compared to the control. ** p < 0.05 compared to the value of TNF-alone. *** p < 0.05 compared to the value of TNF-␣ with rapamycin.
tions such as proliferation and cell survival in a variety of cells (Coffer et al., 1998). Akt is activated by the phosphorylation of serine residues (Coffer et al., 1998). In this study, rapamycin enhanced the TNF-␣-induced phosphorylation of both p44/p42 MAP kinase and Akt in osteoblast-like MC3T3-E1 cells. In addition, we found that Akt inhibitor (Hu et al., 2000) not only suppressed the TNF␣-stimulated IL-6 synthesis but also reduced the amplification by rapamycin of the TNF-␣-stimulated IL-6 synthesis. Moreover, PD98059 (Alessi et al., 1995) as well as Akt inhibitor, reduced the rapamycin-induced enhancement in TNF-␣-stimulated synthesis of IL-6. Taken together, these findings provides compelling evidence that p70 S6 kinase negatively regulates the TNF-␣stimulated IL-6 synthesis at a point upstream of p44/p42 MAP kinase and Akt in osteoblast-like MC3T3-E1 cells. We also found that rapamycin enhanced the IL-6 synthesis in addition to the phosphorylation of Akt induced by TNF-␣ in NHOst, a different osteoblastic cell type. Therefore, it is likely that negative regulation by p70 S6K of the TNF-␣-stimulated IL-6 synthesis through Akt is common in osteoblasts. The potential mechanism of regulation by p70 S6 kinase in TNF-␣-stimulated IL-6 synthesis in osteoblasts shown here is summarized in Fig. 6. It has been shown that TNF-␣ increases the expression of IL-6 gene at least in part
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through activation of nuclear factor-B (NF-B) in osteoblast-like cells (Kurokouchi et al., 1998). In addition, tumor necrosis factor receptor (TNFR) 2 reportedly facilitates phosphatidylinositol 3-kinase-dependent NF-B activation (Marchetti et al., 2004). It might be possible that NF-B activation is also regulated by p70 S6 kinase in osteoblasts. Further investigation would be required to clarify the details. It is generally known that the inflammatory cytokines such as IL-1, IL-6 and TNF-␣ secreted from osteoblasts play pivotal roles in the pathogenesis of postmenopausal osteoporosis through upregulating osteoclasts recruitment and their activities (Manolagas and Jilka, 1995). In addition, over-expression of inflammatory cytokines is well known to be involved in the important clinical feature of inflammatory arthritis including rheumatoid arthritis (Deleuren et al., 1992; Gravallese et al., 2000; Lam et al., 2000; Shigeyama et al., 2000; Wei et al., 2005). In the last decade, monoclonal antibody against TNF-␣ such as infliximab has shown clinical efficacy in controlling the inflammatory signs and symptoms of rheumatoid arthritis, indicating the importance of TNF-␣-signaling in the pathogenesis. Our present findings, indicating that the p70 S6 kinase signaling activated by TNF-␣ limits the TNF-␣-induced oversynthesis of IL-6 in osteoblast-like MC3T3-E1 cells, might provide a new aspect of therapeutics for both metabolic and inflammatory bone diseases. The adequate regulation of the p70 S6 kinase pathway in osteoblasts, as well as the TNF-␣ pathway, could be a new strategy of pathological bone resorption concurrent with these diseases. In conclusion, our results strongly suggest that p70 S6 kinase negatively regulates TNF-␣-induced IL-6 synthesis at a point upstream of p44/p42 MAP kinase and Akt in osteoblast cell lines. Further research to confirm this in osteoblasts themselves is needed. Acknowledgements We are very grateful to Yoko Kawamura for her skillful technical assistance. This investigation was supported in part by Grant-in-Aid for Scientific Research (16590873 and 16591482) for the Ministry of Education, Science, Sports and Culture of Japan, the Foundation for Growth Science, Health and Labour Sciences, the Research Grants for Research on Proteomics and Research on Longevity Sciences from the Ministry of Health, Labour and Welfare of Japan. References
Fig. 7. Schematic representation of the involvement of p70 S6 kinase in the mechanism of the TNF-␣-stimulated IL-6 synthesis in osteoblast-like MC3T3-E1 cells. Abbreviations used are as follows: TNF-␣, tumor necrosis factor-␣; MAP kinase, mitogen-activated protein; IL-6, interleukin-6.
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