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Endocrinology Vol. 140, No. 11 5120-5125
Copyright © 1999 by The Endocrine Society

ARTICLES

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 C1

Masaichi Miwa, Osamu Kozawa, Haruhiko Tokuda and Toshihiko Uematsu

Department of Internal Medicine, Gifu Social Insurance Hospital (M.M.), Kani, Gifu 509-0206; the Department of Pharmacology, Gifu University School of Medicine (O.K., T.U.), Gifu 500-8705; and the Department of Internal Medicine, Chubu National Hospital: National Institute for Longevity Sciences (H.T.), Obu, Aichi 474-8511, Japan

Address all correspondence and requests for reprints to: Dr. Osamu Kozawa, Department of Pharmacology, Gifu University School of Medicine, Gifu 500-8705, Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We previously reported that interleukin-1{alpha} (IL-1{alpha})-induced activation of protein kinase C (PKC) via phosphatidylcholine-specific phospholipase C (PC-PLC) limits IL-6 synthesis induced by IL-1{alpha} itself in osteoblast-like MC3T3-E1 cells. In the present study, we further investigated the mechanism behind IL-1{alpha}-induced IL-6 synthesis in MC3T3-E1 cells. IL-1{alpha} time-dependently stimulated the phosphorylation of both p42/p44 mitogen-activated protein (MAP) kinase and p38 MAP kinase. PD98059, a specific inhibitor of the upstream kinase that activates p42/p44 MAP kinase, inhibited the IL-1{alpha}-induced IL-6 synthesis as well as the phosphorylation of p42/p44 MAP kinase induced by IL-1{alpha}. SB203580, a specific inhibitor of p38 MAP kinase, also reduced both the phosphorylation of p38 MAP kinase and the IL-6 synthesis. 1-Oleoyl-2-acetylglycerol, an activator of PKC, suppressed the IL-1{alpha}-induced IL-6 synthesis. Calphostin C, a specific inhibitor of PKC, or D-609, a specific inhibitor of PC-PLC, significantly enhanced the IL-1{alpha}-induced phosphorylation of p42/p44 MAP kinase without affecting the phosphorylation of p38 MAP kinase. The phosphorylation of p42/p44 MAP kinase by IL-1{alpha} was markedly increased in PKC-down-regulated MC3T3-E1 cells. Neither 12-O-tetradecanoylphorbol-13-acetate, known to be an activator of PKC, nor 1-oleoyl-2-acetylglycerol affected the phosphorylation of p38 MAP kinase induced by IL-1{alpha}. These results strongly suggest that IL-1{alpha}-induced IL-6 synthesis is mediated via activations of both p42/p44 MAP kinase and p38 MAP kinase in osteoblasts, and that PKC activated by IL-1{alpha} itself negatively regulates IL-6 synthesis at a point upstream from p42/p44 MAP kinase.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INTERLEUKIN-1 (IL-1) is known as one of multifunctional cytokines responsible for inflammation, infection, and cancer, and it induces numerous physiological effects on a wide variety of cells (1, 2). In bone, it is well recognized that IL-1 is a potent bone-resorptive agent (3, 4). Bone metabolism is maintained by two types of functional bone cells, osteoblasts and osteoclasts, which are responsible for bone formation and bone resorption, respectively (3). Accumulating evidence indicates that osteoblasts, rather than osteoclasts, possess receptors for many bone-resorptive agents (3). It has been reported that IL-1{alpha} as well as other bone-resorptive agents, such as PTH, tumor necrosis factor-{alpha}, and platelet-derived growth factor, stimulate the production of IL-6 in cultured osteoblasts (4, 5, 6, 7). As IL-6 has been shown to stimulate bone resorption and induce osteoclast formation (4, 8), it is currently recognized that IL-6 secreted from osteoblasts plays a key role as an important downstream effector of bone-resorptive agents. We have recently reported that IL-1{alpha} activates protein kinase C (PKC) through phosphatidylcholine hydrolysis by phosphatidylcholine-specific phospholipase C (PC-PLC) in osteoblast-like MC3T3-E1 cells, and that the PKC activation then limits IL-6 synthesis induced by IL-1{alpha} itself (9). However, the stimulative signaling pathway underlying the IL-1-induced IL-6 synthesis in osteoblasts has not yet been clarified.

The mitogen-activated protein (MAP) kinase family plays important roles in intracellular signaling of a variety of agonists (10). It is well known that MAP kinases are activated on dual phosphorylation of threonine and tyrosine (10). In osteoblastic cells, MAP kinases have been reported to be involved in various intracellular signaling pathways (11, 12, 13, 14, 15). As for intracellular signaling of IL-1, IL-1 has been shown to activate p42/p44 MAP kinase in rabbit articular chondrocytes (16). In addition, it has recently been reported that p38 MAP kinase is involved in IL-1ß-induced IL-6 gene expression in human fibroblast-like synoviocytes (17). Furthermore, IL-1ß reportedly activates extracellular signal-regulated kinase, the so-called p42/p44 MAP kinase, and c-Jun NH2-terminal kinase pathways in normal human osteoblastic cells and rat osteosarcoma UMR-106 cells (15). Thus, the present study was conducted to clarify whether p42/p44 MAP kinase and/or p38 MAP kinase are involved in the IL-1{alpha}-induced IL-6 synthesis in osteoblast-like MC3T3-E1 cells. We here show that IL-1{alpha}-induced IL-6 synthesis is mediated via activations of both p42/p44 MAP kinase and p38 MAP kinase in these cells, and that the PKC activation negatively regulates IL-6 synthesis at a point upstream from p42/p44 MAP kinase.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
IL-1{alpha} and potassium tricyclo-(5,2,1,0)-decyl-[9(8)-xanthogenate] (D-609) were obtained from Funakoshi Pharmaceutical Co. (Tokyo, Japan). 2'-Amino-3'-methoxyflanone (PD98059) and 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)IH-imidazole (SB203580) were obtained from Calbiochem (La Jolla, CA). Calphostin C and 12-O-tetradecanoylphorbol-13-acetate (TPA) were purchased from Sigma Chemical Co. (St. Louis, MO). 1-Oleoyl-2-acetylglycerol (OAG) was purchased from Nacalai Tesque (Kyoto, Japan). Phospho-specific p42/p44 MAP kinase antibodies (rabbit polyclonal IgG, affinity purified), p42/p44 MAP kinase antibodies (rabbit polyclonal IgG, affinity purified), phospho-specific p38 MAP kinase antibodies (rabbit polyclonal IgG, affinity purified), and p38 MAP kinase antibodies (rabbit polyclonal IgG, affinity purified) were purchased from New England Biolabs, Inc. (Beverly, MA). The mouse IL-6 enzyme immunoassay kit, the enhanced chemiluminescence Western blotting detection system, the PKC assay system, and [{gamma}-32P]ATP (3000 Ci/mmol) were purchased from Amersham Pharmacia Biotech Japan (Tokyo, Japan). Other materials and chemicals were obtained from commercial sources. PD98059, SB203580, calphostin C, and TPA were dissolved in dimethylsulfoxide. The maximal concentration of dimethylsulfoxide was 0.1%, which did not affect the assay for IL-6 or the analyses of MAP kinases.

Cell culture
Cloned osteoblast-like MC3T3-E1 cells derived from newborn mouse calvaria (18, 19) were maintained as previously described (20). Briefly, the cells were cultured in {alpha}MEM containing 10% FCS at 37 C in a humidified atmosphere of 5% CO2-95% air. The cells (5 x 104) were seeded into 35-mm diameter dishes in 2 ml {alpha}MEM containing 10% FCS. After 5 days, the medium was exchanged for 2 ml {alpha}MEM containing 0.3% FCS. The cells were used for experiments after 48 h. When indicated, the cells were pretreated with 0.1 µM TPA for 24 h, as previously reported (21).

Assay for IL-6
The cultured cells were pretreated with various doses of PD98059, SB203580, or OAG for 20 min, and then stimulated by 30 ng/ml (~1.66 nM) IL-1{alpha} or vehicle in 1 ml {alpha}MEM containing 0.3% FCS for the indicated periods. The conditioned medium was collected, and IL-6 in the medium was measured by an IL-6 enzyme immunoassay kit.

Analyses of MAP kinases
The cultured cells were stimulated by IL-1{alpha} in 1 ml {alpha}MEM for the indicated periods. The cells were washed twice with 1 ml PBS and then lysed, homogenized, and sonicated in a lysis buffer containing 62.5 mM Tris/HCl (pH 6.8), 2% SDS, 50 mM dithiothreitol, and 10% glycerol. The cytosolic fraction was collected as a supernatant after centrifugation at 125,000 x g for 10 min at 4 C. SDS-PAGE was performed by the method of Laemmli (22) in 10% polyacrylamide gel. Western blotting analysis was performed as described previously (23) using phospho-specific p42/p44 MAP kinase antibodies, p42/p44 MAP kinase antibodies, phospho-specific p38 MAP kinase antibodies or p38 MAP kinase antibodies, and peroxidase-labeled antibodies raised in goat against rabbit IgG as second antibodies. Peroxidase activity on the nitrocellulose sheet was visualized on x-ray film using the enhanced chemiluminescence Western blotting detection system. When indicated, the cells were pretreated with PD98059, SB203580, calphostin C, D-609, TPA, or OAG for 20 min.

Assay for PKC activity
The cultured cells were pretreated with calphostin C for 20 min and then stimulated by IL-1{alpha} at 37 C for 10 min in 1 ml assay buffer [5 mM HEPES (pH 7.4), 150 mM NaCl, 5 mM KCl, 5.5 mM glucose, 0.8 mM MgSO4, and 1 mM CaCl2] containing 0.01% BSA. The reaction was terminated by aspirating the medium, and PKC activity was determined using a PKC assay system.

Statistical analysis
The data were analyzed by one-way ANOVA, followed by Bonferroni’s method for multiple comparison between pairs. P < 0.05 was considered significant. All data are presented as the mean ± SEM of triplicate determinations. Each experiment was repeated three times with similar results.

Determinations
The absorbance of enzyme immunoassay samples was measured at 450 nm with an EL 340 Bio Kinetic Reader (Bio-Tek Instruments, Inc., Winooski, VT). The radioactivity of 32P samples for PKC activity was determined with an LS-6500IC liquid scintillation spectrometer (Beckman Coulter, Inc., Palo Alto, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of IL-1{alpha} on the phosphorylation of p42/p44 MAP kinase or p38 MAP kinase in MC3T3-E1 cells
To investigate whether IL-1{alpha} activates p42/p44 MAP kinase and/or p38 MAP kinase in osteoblast-like MC3T3-E1 cells, we examined the effect of IL-1{alpha} on the phosphorylation of p42/p44 MAP kinase and p38 MAP kinase by Western blotting analysis. IL-1{alpha} time dependently elicited the phosphorylation of p42/p44 MAP kinase and p38 MAP kinase (Fig. 1Go). The phosphorylation of both p42/p44 MAP kinase and p38 MAP kinase were detectable for 3–10 min after the stimulation of IL-1{alpha}. The maximal effects of IL-1{alpha} on the phosphorylation of p42/p44 MAP kinase and p38 MAP kinase were observed at 10 and 5 min, respectively (Fig. 1Go). Despite using the antibodies that recognize both p42 and p44 MAP kinases, the band of p44 MAP kinase appeared much fainter than that of p42 MAP kinase in MC3T3-E1 cells.



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  		Figure 1. Time-dependent effect of IL-1{alpha} on the phosphorylation of MAP kinases in MC3T3-E1 cells. The cultured cells were stimulated by 30 ng/ml IL-1{alpha} (lanes 2–7) or vehicle (lanes 1 and 8) for the indicated periods. A, p42/p44 MAP kinase. Upper panel, Antiphospho-specific p42/p44 MAP kinase antibodies. Lower panel, Anti-p42/p44 MAP kinase antibodies. B, p38 MAP kinase. Upper panel, Antiphospho-specific p38 MAP kinase antibodies. Lower panel, Anti-p38 MAP kinase antibodies. The results are representative of triplicate independent experiments.

 
Effect of PD98059 or SB203580 on IL-1{alpha}-induced IL-6 synthesis in MC3T3-E1 cells
We previously showed that IL-6 synthesis is stimulated by IL-1{alpha} in osteoblast-like MC3T3-E1 cells (9). To clarify whether the activation(s) of p42/p44 MAP kinase and/or p38 MAP kinase is involved in IL-1{alpha}-induced IL-6 synthesis in these cells, we examined the effect of PD98059, a specific inhibitor of the upstream kinase that activates p42/p44 MAP kinase (24, 25), or SB203580, an inhibitor of p38 MAP kinase (26, 27), on the IL-6 synthesis induced by IL-1{alpha}. As previously reported (9), IL-1{alpha} stimulated IL-6 synthesis in a time-dependent manner up to 24 h. PD98059, which alone had little effect on IL-6 synthesis, significantly inhibited IL-1{alpha}-induced IL-6 synthesis (Fig. 2AGo). The inhibitory effect of PD98059 on IL-1{alpha}-induced IL-6 synthesis was dose dependent in the range between 0.1–50 µM (Fig. 2BGo). The maximal inhibitory effect of PD98059 was achieved at 20 µM, a dose that led to about 75% reduction of the IL-1{alpha} effect, and this effect was not statistically different from that of 50 µM. In addition, SB203580, which alone hardly affected the basal level of IL-6 synthesis, markedly reduced IL-1{alpha}-induced IL-6 synthesis (Fig. 3AGo). The inhibitory effect of SB203580 on IL-1{alpha}-induced IL-6 synthesis was dose dependent in the range between 0.1–30 µM (Fig. 3BGo). The maximal inhibitory effect of SB203580 was observed at 30 µM, a dose that led to an almost complete reduction of the IL-1{alpha} effect. We confirmed that PD98059 reduced the IL-1{alpha}-induced phosphorylation of p42/p44 MAP kinase (Fig. 4AGo), and that SB203580 suppressed the phosphorylation of p38 MAP kinase induced by IL-1{alpha} in these cells (Fig. 4BGo).



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  		Figure 2. Effect of PD98059 on IL-1{alpha}-induced IL-6 synthesis in MC3T3-E1 cells. A, The cultured cells were pretreated with 20 µM PD98059 ({blacktriangleup} and {triangleup}) or vehicle (• and {circ}) for 20 min, and then stimulated by 30 ng/ml IL-1{alpha} ({blacktriangleup} and •) or vehicle ({triangleup} and {circ}) for the indicated periods. B, The cultured cells were pretreated with various doses of PD98059 for 20 min, and then stimulated by 30 ng/ml IL-1{alpha} (•) or vehicle ({circ}) for 24 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 with the value of IL-1{alpha} alone.

 


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  		Figure 3. Effect of SB203580 on IL-1{alpha}-induced IL-6 synthesis in MC3T3-E1 cells. A, The cultured cells were pretreated with 3 µM SB203580 ({blacktriangleup} and {triangleup}) or vehicle (• and {circ}) for 20 min, and then stimulated by 30 ng/ml IL-1{alpha} ({blacktriangleup} and •) or vehicle ({triangleup} and {circ}) for the indicated periods. B, The cultured cells were pretreated with various doses of SB203580 for 20 min, and then stimulated by 30 ng/ml IL-1{alpha} (•) or vehicle ({circ}) for 24 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 with the value of IL-1{alpha} alone.

 


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  		Figure 4. Effects of PD98059 or SB203850 on the IL-1{alpha}-induced phosphorylation of p42/p44 MAP kinase and p38 MAP kinase in MC3T3-E1 cells. A, The cultured cells were pretreated with 20 µM PD98059 or vehicle for 20 min, and then stimulated by 30 ng/ml IL-1{alpha} or vehicle (lanes 1, 3, and 5) for 5 min. Upper panel, Antiphospho-specific p42/p44 MAP kinase antibodies. Lower panel, Anti-p42/p44 MAP kinase antibodies. B, The cultured cells were pretreated with 3 µM SB203580 or vehicle for 20 min, and then stimulated by 30 ng/ml IL-1{alpha} or for 5 min. Upper panel, Antiphospho-specific p38 MAP kinase antibodies. Lower panel, Anti-p38 MAP kinase antibodies. The results are representative of triplicate independent experiments.

 
Effect of calphostin C or D-609 on IL-1{alpha}-induced phosphorylation of p42/p44 MAP kinase or p38 MAP kinase in MC3T3-E1 cells
We demonstrated that IL-1{alpha} induces phosphatidylcholine hydrolysis by PC-PLC, resulting in the activation of PKC in osteoblast-like MC3T3-E1 cells, and that the PKC activation then limits IL-6 synthesis induced by IL-1{alpha} itself (9). In addition, we confirmed that OAG, a synthetic diacylglycerol known to be an activator of PKC (28), which alone had little effect on the synthesis of IL-6, suppressed IL-1{alpha}-induced IL-6 synthesis (Table 1Go). To investigate whether the limitation of IL-1{alpha}-induced IL-6 synthesis by PKC is exerted in the p42/p44 MAP kinase- and/or p38 MAP kinase-mediating pathway(s), we examined the effect of calphostin C, a highly potent and specific inhibitor of PKC (29), on the IL-1{alpha}-induced phosphorylation of p42/p44 MAP kinase or p38 MAP kinase. Calphostin C, which alone weakly reduced basal phosphorylation of p42/p44 MAP kinase, significantly enhanced IL-1{alpha}-induced p42/p44 MAP kinase phosphorylation (Fig. 5AGo). However, calphostin C, which by itself hardly affected the phosphorylation of p38 MAP kinase, had little effect on IL-1{alpha}-induced p38 MAP kinase phosphorylation (Fig. 5BGo). We found that calphostin C suppressed IL-1{alpha}-induced PKC activation in MC3T3-E1 cells to about 10% of that in untreated cells (Fig. 6Go). In the recent study (9) we showed that calphostin C enhances IL-1{alpha}-induced IL-6 synthesis in these cells. In addition, we previously demonstrated that D-609, a specific inhibitor of PC-PLC (30), amplifies the IL-6 synthesis induced by IL-1{alpha} in MC3T3-E1 cells (9). Thus, we next examined the effect of D-609 on the phosphorylation of p42/p44 MAP kinase or p38 MAP kinase in these cells. D-609, which by itself weakly suppressed basal phosphorylation of p42/p44 MAP kinase but hardly affected that of p38 MAP kinase, significantly amplified IL-1{alpha}-induced p42/p44 MAP kinase phosphorylation without affecting the phosphorylation of p38 MAP kinase induced by IL-1{alpha} (Fig. 5Go).


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  		Table 1. Effect of OAG on IL-1{alpha}-induced IL-6 synthesis in MC3T3-E1 cells

 


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  		Figure 5. Effects of calphostin C or D-609 on the IL-1{alpha}-induced phosphorylation of p42/p44 MAP kinase and p38 MAP kinase in MC3T3-E1 cells. The cultured cells were pretreated with 0.1 µM calphostin C (CalC; lanes 3 and 4), 30 ng/ml D-609 (lanes 5 and 6), or vehicle (Veh; lanes 1 and 2) for 20 min, and then stimulated by 30 ng/ml IL-1{alpha} (lanes 2, 4, and 6) or vehicle (lanes 1, 3, and 5) for 5 min. A, p42/44 MAP kinase. Upper panel, Antiphospho-specific p42/p44 MAP kinase antibodies. Lower panel, Anti-p42/p44 MAP kinase antibodies. B, p38 MAP kinase. Upper panel, Antiphospho-specific p38 MAP kinase antibodies. Lower panel, Anti-p38 MAP kinase antibodies. The results are representative of triplicate independent experiments.

 


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  		Figure 6. Effect of calphostin C on the IL-1{alpha}-induced PKC activation in MC3T3-E1 cells. The cultured cells were pretreated with 0.1 µM calphostin C (CalC) or vehicle for 20 min, and then stimulated by 30 ng/ml IL-1{alpha} or vehicle for 10 min. Each value represents the mean ± SEM of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *, P < 0.05 compared with the value of IL-1{alpha} alone.

 
Effect of PKC down-regulation on IL-1{alpha}-induced phosphorylation of p42/p44 MAP kinase in MC3T3-E1 cells
In a recent study (9) we showed that the IL-1{alpha}-induced IL-6 synthesis is amplified in PKC down-regulated MC3T3-E1 cells by long term pretreatment with TPA, which is known to be a direct activator of PKC (28). Thus, we next examined the effect of PKC down-regulation on the IL-1{alpha}-induced p42/p44 MAP kinase phosphorylation in these cells. We previously confirmed that the binding capacity of [3H]phorbol 12,13-dibutyrate, a PKC-activating phorbol ester (28), in TPA (0.1 µM for 24 h)-pretreated cells is reduced to approximately 30% of the capacity in intact cells (31). This result suggests that PKC down-regulation truly occurs in MC3T3-E1 cells with long term TPA treatment. The long term pretreatment with TPA, which by itself had no effect on the phosphorylation of p42/p44 MAP kinase, markedly increased the IL-1{alpha}-induced phosphorylation of p42/p44 MAP kinase compared with that in cells without TPA treatment (Fig. 7Go).



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  		Figure 7. Effect of long-term TPA pretreatment on the IL-1{alpha}-induced phosphorylation of p42/p44 MAP kinase in MC3T3-E1 cell. The cultured cells were pretreated with 0.1 µM TPA (lanes 3 and 4) or vehicle (lanes 1 and 2) for 24 h, and then stimulated by 30 ng/ml IL-1{alpha} (lanes 2 and 4) or vehicle (lanes 1 and 3) for 5 min. Upper panel, Antiphospho-specific p42/p44 MAP kinase antibodies. Lower panel, Anti-p42/p44 MAP kinase antibodies. The results are representative of triplicate independent experiments.

 
Effects of TPA or OAG on IL-1{alpha}-induced phosphorylation of p38 MAP kinase in MC3T3-E1 cells
To elucidate whether the exogenously activated PKC affects the phosphorylation of p38 MAP kinase, we further examined the effect of TPA or OAG on the IL-1{alpha}-induced p38 MAP kinase phosphorylation in MC3T3-E1 cells. Either TPA or OAG, which alone had no effect on the phosphorylation of p38 MAP kinase, failed to affect the IL-1{alpha}-induced phosphorylation of p38 MAP kinase (Fig. 8Go).



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  		Figure 8. Effects of short term pretreatment with TPA or OAG on the IL-1{alpha}-induced phosphorylation of p38 MAP kinase in MC3T3-E1 cell. The cultured cells were pretreated with 0.1 µM TPA (lanes 3 and 4), 0.1 mM OAG (lanes 5 and 6), or vehicle (lanes 1 and 2) for 20 min, and then stimulated by 30 ng/ml IL-1{alpha} (lanes 2, 4, and 6) or vehicle (lanes 1, 3, and 5) for 5 min. Upper panel, Antiphospho-specific p38 MAP kinase antibodies. Lower panel, Anti-p38 MAP kinase antibodies. The results are representative of triplicate independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we demonstrated that the phosphorylation of both p42/p44 MAP kinase and p38 MAP kinase was elicited by IL-1{alpha} in osteoblast-like MC3T3-E1 cells. It is well known that MAP kinases are activated by phosphorylation of threonine and tyrosine residues by dual specificity MAP kinase kinase (10, 32). Therefore, these findings suggest that IL-1{alpha} activates both p42/p44 MAP kinase and p38 MAP kinase in these cells. In addition, we showed that both PD98059 and SB203580 markedly suppressed the IL-1{alpha}-induced IL-6 synthesis. We also found that the IL-1{alpha}-induced phosphorylation of p42/p44 MAP kinase and p38 MAP kinase was truly inhibited by PD98059 and SB203580, respectively, in these cells. Based on our present results, it is most likely that the activations of both p42/p44 MAP kinase and p38 MAP kinase are involved in the IL-6 synthesis induced by IL-1{alpha} in osteoblast-like MC3T3-E1 cells.

In a recent study (9) we demonstrated that IL-1{alpha}-activated PKC, through phosphatidylcholine hydrolysis by PC-PLC, negatively regulates IL-6 synthesis by IL-1{alpha} itself in osteoblast-like MC3T3-E1 cells. We here confirmed that OAG suppressed the IL-1{alpha}-induced IL-6 synthesis in these cells. Thus, the detailed regulatory mechanism of PKC in the IL-6 synthesis induced by IL-1{alpha} was investigated. We showed that calphostin C, which hardly affected the phosphorylation of p38 MAP kinase induced by IL-1{alpha}, significantly enhanced the phosphorylation of p42/p44 MAP kinase induced by IL-1{alpha} in these cells. Thus, it seems that PKC activated by IL-1{alpha} inhibited the activation of p42/p44 MAP kinase induced by IL-1{alpha} without affecting IL-1{alpha}-induced activation of p38 MAP kinase. In addition, we demonstrated that D-609 amplified the IL-1{alpha}-induced phosphorylation of p42/p44 MAP kinase without affecting the IL-1{alpha}-induced phosphorylation of p38 MAP kinase. Thus, these results suggest that the IL-1{alpha}-activated PKC through PC-PLC suppresses the IL-1{alpha}-induced activation of p42/p44 MAP kinase. Taking our findings into account, it is most likely that p42/p44 MAP kinase, not p38 MAP kinase, is regulated by PKC in the intracellular signaling elicited by IL-1{alpha} in MC3T3-E1 cells, and that the attenuation of p42/p44 MAP kinase activation by IL-1{alpha} results in suppressing the oversynthesis of IL-6. Furthermore, we showed that the IL-1{alpha}-induced phosphorylation of p42/p44 MAP kinase was markedly increased in the PKC down-regulated cells, suggesting that the inhibitory effect of PKC is exerted at a point upstream from p42/p44 MAP kinase. Thus, it is probable that our previous results showing the enhancement of IL-1{alpha}-induced IL-6 synthesis in PKC down-regulated MC3T3-E1 cells mainly results from the amplification of p42/p44 MAP kinase activation. In general, it is most likely that the limitation of IL-1{alpha}-induced IL-6 synthesis by PKC activation through PC-PLC is exerted at a point upstream from p42/p44 MAP kinase in osteoblast-like MC3T3-E1 cells. It has recently been reported that PGF2{alpha} activates p42/p44 MAP kinase through PKC in these cells (33). Thus, it is probable that PKC possesses dual effects as an activator and a suppressor of p42/p44 MAP kinase in these cells, and these effects are dependent upon each agonist.

In addition, we showed that TPA or OAG had little effect on IL-1{alpha}-induced phosphorylation of p38 MAP kinase in MC3T3-E1 cells. Therefore, it seems unlikely that the PKC activation modulates IL-1{alpha}-induced activation of p38 MAP kinase in osteoblast-like MC3T3-E1 cells. It has recently been reported that p38 MAP kinase is involved in IL-1ß-induced IL-6 gene expression in human fibroblast-like synoviocytes (17). From our findings, it is probable that p38 MAP kinase also acts as a stimulative regulator in IL-1{alpha}-induced IL-6 synthesis in osteoblasts.

In conclusion, our present results strongly suggest that IL-1{alpha}-induced IL-6 synthesis is mediated via activations of both p42/p44 MAP kinase and p38 MAP kinase in osteoblasts, and that PKC activation negatively regulates IL-6 synthesis at a point upstream from p42/p44 MAP kinase.


   Acknowledgments
 
We are very grateful to Hidenori Kawamura and Daijiro Hatakeyama for their skillful technical assistance.


   Footnotes
 
1 This work was supported in part by a Grant-in-Aid for Scientific Research (09671041) from the Ministry of Education, Science, Sports, and Culture of Japan. Back

Received January 11, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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