Basic Fibroblast Growth Factor Induces Interleukin-6 Synthesis in Osteoblasts: Autoregulation by Protein Kinase C

Cell. Signal. Vol. 9, No. 6, pp. 463–468, 1997 Copyright  1997 Elsevier Science Inc.

ISSN 0898-6568/97 $17.00 PII S0898-6568(97)00043-0

Basic Fibroblast Growth Factor Induces Interleukin-6 Synthesis in Osteoblasts: Autoregulation by Protein Kinase C Osamu Kozawa,†* Atsushi Suzuki‡ and Toshihiko Uematsu† †Department of Pharmacology, Gifu University School of Medicine, Gifu 500, Japan and ‡First Department of Internal Medicine, Nagoya University School of Medicine, Nagoya 466, Japan

ABSTRACT. We previously reported that basic fibroblast growth factor (bFGF) stimulates both phospholipases C and D via independent pathways in osteoblastlike MC3T3-E1 cells. In this study, we investigated the effect of bFGF on interleukin-6 (IL-6) synthesis in these cells. bFGF stimulated the IL-6 synthesis dose-dependently in the range between 1 and 30 ng/ml. The depletion of extracellular Ca21 by EGTA suppressed the bFGFinduced IL-6 synthesis. TMB-8, an inhibitor of intracellular Ca21 mobilization, also inhibited the IL-6 synthesis by bFGF. bFGF stimulated the Ca21 influx from extracellular space. Genistein, a tyrosine kinase inhibitor, suppressed the bFGF-induced Ca21 influx. Staurosporine, an inhibitor for protein kinases, enhanced the bFGFinduced IL-6 synthesis. Calphostin C, a highly potent and specific inhibitor for protein kinase C (PKC), also enhanced the IL-6 synthesis by bFGF. The bFGF-induced IL-6 synthesis was amplified in PKC down-regulated cells. U-73122, a phospholipase C inhibitor, enhanced the bFGF-induced IL-6 synthesis. Propranolol, a phosphatidic acid phosphohydrolase inhibitor, also enhanced the IL-6 synthesis by bFGF. These results strongly suggest that bFGF stimulates IL-6 synthesis, which depends on intracellular Ca21 mobilization in osteoblastlike cells, and that the IL-6 synthesis by bFGF is autoregulated due to PKC activation. cell signal 9;6:463–468, 1997.  1997 Elsevier Science Inc. KEY WORDS. Interleukin-6, Basic fibroblast growth factor, Protein kinase C, Osteoblast

INTRODUCTION Growth factors, either circulating in blood or locally released, are recognized to be regulators in proliferation and differentiation of ubiquitous cells [1]. It has been reported that large quantities of endogenous growth factors exist also in bone matrix [2]. Basic fibroblast growth factor (bFGF or FGF-2) is found in bone matrix, and cultured osteoblastlike cells produce bFGF [3–5]. In addition, bFGF expression in macrophages, osteoblasts or chondrocytes is detected during all stages of fracture repair: Stage 1, immediate injury response; Stage 2, intramembranous ossification; Stage 3, chondrogenesis; Stage 4, endochondral ossification [6]. Thus, it is nowadays recognized that bFGF plays an important role in bone metabolism and fracture healing. It has been reported that bFGF stimulates proliferation of osteoblasts [5], and that bFGF decreases insulinlike growth factor-I synthesis in rat calvaria cultures, reduces markers of differentiation of osteoblasts such as alkaline phosphatase activity, osteocalcin mRNA level and type I collagen mRNA level in osteoblasts including osteoblastlike MC3T3-E1 cells [7–9]. As for *Author to whom all correspondence should be addressed. Abbreviations: a-MEM–a-minimum essential medium; FCS–fetal calf serum; FGF–fibroblast growth factor; PL–phospholipase; PKC–protein kinase C; TPA–12-O-tetradecanoylphorbol-13-acetate. Received 3 December 1996; and accepted 1 February 1997.

intracellular signalling system of bFGF, there are four structurally related high-affinity receptors (FGF receptor 1 to 4), which have an intrinsic protein tyrosine kinase activity, and which elicit tyrosine autophosphorylation of the receptors [10, 11]. In a previous study [12], we have shown that bFGF autophosphorylates FGF receptor 1 and 2 in MC3T3E1 cells. It has been reported that phospholipase (PL) Cg is activated following tyrosine phosphorylation and that bFGF stimulates phosphoinositide hydrolysis by PLCg [13, 14]. Phosphoinositide hydrolysis by PLC results in the formation of two second messengers, diacyglycerol, which activates protein kinase C (PKC), and inositol trisphosphate, which mobilizes intracellular Ca 21 [15, 16]. We have recently reported that bFGF induces both phosphoinositide hydrolysis by PLC and phosphatidylcholine hydrolysis by PLD in osteoblastlike MC3T3-E1 cells [12]. Interleukin (IL)-6 is well-known to be a multifunctional cytokine that is involved in the regulation of the immune response, hematopoiesis, and inflammation [17, 18]. In bone metabolism, it has been reported that IL-6 is produced by bone resorptive agents such as parathyroid hormone, IL-1, TNF and platelet-derived growth factor in osteoblasts including osteoblastlike MC3T3-E1 cells [19–22]. In addition, IL-6 has been reported to increase the formation of osteoblasts from hematopoietic progenitors [20] and to enhance osteo-

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clast formation in foetal calvariae [21]. So, IL-6 is now recognized to be an important osteotropic agent as an autocrine/paracrine factor in bone metabolism [23]. However, the precise mechanism of IL-6 synthesis in osteoblasts has not yet been fully clarified. In the present study, we examined the effect of bFGF on IL-6 synthesis in osteoblastlike MC3T3-E1 cells. We here show that bFGF stimulates IL-6 synthesis, which depends on intracellular Ca21 mobilization in these cells, and that self-induced PKC activation by both PLC and PLD has an inhibitory effect on the IL-6 synthesis. MATERIALS AND METHODS Materials

O. Kozawa et al.

Assay for 45Ca21 Influx Assay for 45Ca21 influx was performed as previously described [26]. In brief, the cultured cells were pretreated with 0.1 mM nifedipine for 10 min in 1 ml of an assay buffer [5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.4, 150 mM NaCl, 5 mM KCl, 5.5 mM glucose, 0.8 mM MgSO4 and 1 mM CaCl2] containing 0.01% bovine serum albumin. The cells were then stimulated by various doses of bFGF containing 5 mCi 45Ca21 at 378C for the indicated periods. After washing 4 times with 1 ml of the cold assay buffer containing EGTA, the reaction was immediately terminated by adding 1 ml of 0.1% sodium dedecyl sulfate. The radioactivity of the lysate was determined. When indicated, the cells were pretreated with genistein for 20 min.

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CaCl2 (10–40 mCi/mg) and mouse IL-6 ELISA system were purchased from Amersham Japan (Tokyo, Japan). bFGF was purchased from Boehringer Mannheim K. K. (Tokyo, Japan). 8-(Diethylamino)octyl-3,4,5,-trimethoxybenzoate hydrochloride (TMB-8), thapsigargin, claphostin C, genistein and 1-(6-((17b-3-methoxyestra-1,3,5(10)-trien17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U-73122) were purchased from Funakoshi Pharmaceutical Co. (Tokyo, Japan). A23187, nifedipine, staurosporine and 12-O-tetradecanoylphorbol-13-acetate (TPA) were purchased from Sigma Chemical Co. (St. Louis, MO). dl-Propranolol hydrochloride (propranolol) was purchased from Wako Pure Chemical Co. (Osaka, Japan). Thapsigargin, A23187, nifedipine, genistein, staurosporine, calphostin C, TPA, U-73122 and propranolol were dissolved in dimethyl sulfoxide. The maximum concentration of dimethyl sulfoxide was 0.1% and this did not affect the assay for IL-6 synthesis nor 45Ca21 influx. Cell Culture Cloned osteoblastlike MC3T3-E1 cells derived from newborn mouse calvaria [24, 25] were generously provided by Dr. M. Kumegawa (Meikai University, Sakado, Japan) and maintained in a-minimum essential medium (a-MEM) containing 10% foetal calf serum (FCS) at 378C in a humidified atmosphere of 5% CO2/95% air. The cells (5 3 104) were seeded into 35-mm diameter dishes in 2 ml of a-MEM containing 10% FCS. After 5 d, the medium was exchanged for 2 ml of a-MEM containing 0.3% FCS. The cells were used for experiments after 48 h. When indicated, the cells were pretreated with 0.1 mM TPA for 24 h. Assay for IL-6 Synthesis The cultured cells were stimulated by bFGF or thapsigargin in 1 ml of a-MEM containing 0.3% FCS for the indicated periods. When indicated, the cells were stimulated by A23187 for 1 h, and subsequently incubated for 48 h. The conditioned medium was collected, and IL-6 in the medium was measured by an IL-6 ELISA kit. When indicated, the cells were pretreated with TMB-8, staurosporine, calphostin C, U-73122 or propranolol for 20 min.

Determination The absorbance of enzyme immunoassay samples was measured at 450 nm with SLT-Labinstruments EAR 340 AT. The radioactivity of 45Ca-samples was determined with a Beckman LS-6500IC liquid scintillation spectrometer. Statistical Analysis The data were analysed by Student’s t-test and a P , 0.05 was considered significant. All data are presented as the mean 6 S. D. of triplicate determinations. RESULTS Effect of bFGF on IL-6 Synthesis in MC3T3-E1 Cells bFGF stimulated the IL-6 synthesis time-dependently up to 48 h in osteoblastlike MC3T3-E1 cells (Fig. 1). The stimulative effect of bFGF was dose-dependent in the range between 1 and 30 ng/ml (Fig. 2). The maximum effect of bFGF on IL-6 synthesis was observed at 30 ng/ml. Effects of Depletion of Extracellular Ca21 and TMB-8 on bFGF-Induced IL-6 Synthesis in MC3T3-E1 cells To clarify the role of intracellular Ca 21 mobilization on bFGF-induced IL-6 synthesis in MC3T3-E1 cells, we examined the effects of the depletion of extracellular Ca21 by EGTA and TMB-8 on the IL-6 synthesis. The bFGFinduced IL-6 synthesis was significantly reduced by chelating extracellular Ca21 by EGTA, which alone did not affect the basal level of IL-6 (Table 1). TMB-8, known to inhibit the intracellular Ca 21 mobilization [27, 28], which by itself had no effect on IL-6 synthesis, also suppressed the IL-6 synthesis by bFGF (Table 1). Thapsigargin, which is known to stimulate Ca21 mobiliztion from intracellular stores [29], markedly induced IL-6 synthesis in MC3T3-E1 cells (12 6 3 pg/ml for control; 720 6 55 pg/ml for 0.1 mM thapsigargin; these as measured during an incubation for 48 h). A23187, a Ca-ionophore, also stimulated IL-6 synthesis in these cells (13 6 2 pg/ml for control; 480 6 38 pg/ml for 1 mM A23187; these as measured during an incubation for 48 h).

IL-6 Synthesis by bFGF

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FIGURE 1. Effect of bFGF on the synthesis of IL-6 in MC3T3-

E1 cells: the time-course of the incubation periods. The cultured cells were stimulated by 30 ng/ml of bFGF (d) or vehicle (s) for the indicated periods. Each value represents the mean 6 S. D. of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *P , 0.05 vs. control value.

Effect of bFGF on 45 Ca21 Influx in MC3T3-E1 Cells 45

21

bFGF stimulated Ca influx, even in the presence of 0.1 mM nifedipine, a Ca21 antagonist that inhibits voltagedependent Ca21 channel [30], time-dependently up to 20 min in MC3T3-E1 cells (Fig. 3A). The stimulative effect of bFGF was dose-dependent in the range between 1 and 30 ng/ml (Fig. 3B). The maximum effect of bFGF on Ca 21 influx was observed at 30 ng/ml. Genistein, an inhibitor for protein tyrosine kinases [31], inhibited the bFGF-induced 45 Ca21 influx dose-dependently in the range between 1 and 30 g/ml (Fig. 4).

Effects of Staurosporine, Calphostin C and Down-regulation of PKC on bFGF-induced IL-6 Synthesis in MC3T3-E1 Cells Staurosporine (10 nM), an inhibitor for protein kinases [32], which alone had no effect on IL-6 synthesis, significantly enhanced the bFGF-induced IL-6 synthesis in MC3T3-E1 cells (Table 2). Calphostin C (0.1 mM), a highly potent and specific inhibitor for PKC [33], which by itself did not affect the basal level of IL-6, also enhanced the IL-6 synthesis by bFGF (Table 2). It has been reported that 24 h pretreatment with 0.1 mM TPA downregulates PKC in osteoblastlike MC3T3-E1 cells [34]. So, we next examined the effect of bFGF on IL-6 synthesis in PKC downregulated cells. The long-term pretreatment with TPA significantly amplified bFGF-induced IL-6 synthesis compared to that in intact cells (Table 3).

FIGURE 2. Dose-dependent effect of bFGF on the synthesis of IL-6 in MC3T3-E1 cells. The cultured cells were stimulated by various doses of bFGF for 48 h. Values for unstimulated cells have been subtracted from each data point. Each value represents the mean 6 S. D. of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *P , 0.05 vs. control value.

Effects of U-73122 and Propranolol on bFGF-induced IL-6 Synthesis in MC3T3-E1 Cells We previously showed that bFGF induces both phosphoinositide hydrolysis by PLC and phosphatidylcholine hydrolysis by PLD via independent pathways in MC3T3-E1 cells [12]. To clarify the role of these two PLs’ activation on IL-6 synthesis by bFGF in MC3T3-E1 cells, we examined the effects of U-73122, an inhibitor for PLC [35], and propranolol, an inhibitor of phosphatidic acid phosphohydrolase [36], on bFGF-induced IL-6 synthesis. The IL-6 synthesis by bFGF was significantly enhanced by U-73122 (10 mM) or propranolol (300 mM), which alone had little effect on the basal level of IL-6 (Table 4). TABLE 1. Effect of EGTA or TMB-8 on bFGF-

induced IL-6 synthesis in MC3T3-E1 cells IL-6 (pg/ml) Control EGTA TMB-8 bFGF bFGF 1 EGTA bFGF 1 TMB-8

12 11 12 185 76 40

6 6 6 6 6 6

3 3 2 16a 10b 8b

The cultured cells were treated with 1.2 mM EGTA or pretreated with 30 mM of TMB-8 or vehicle for 20 min. The cells were then stimulated by 30 ng/ml of bFGF or vehicle for 48 h. Each value represents the mean 6 S.D. of triplicate determinations. Similar results were obtained with two additional and different cell preparations. aP , 0.05 vs. control value. b P , 0.05 vs. value of bFGF alone.

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Ca21 influx in MC3T3-E1 cells. (A) Time course. The cultured cells were stimulated by 30 ng/ml of bFGF (d) or vehicle (s) with 5 mCi 45Ca21 for the indicated periods; (B) Dose-dependency. The cells were stimulated by various doses of bFGF with 5 mCi 45Ca21 for 20 min. Each value represents the mean 6 S. D. of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *P, 0.05 vs. control value. FIGURE 3. Effect of bFGF on

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DISCUSSION In the present study, we showed that bFGF stimulated IL-6 synthesis in osteoblastlike MC3T3-E1 cells. As far as we know it is probably the first report showing that bFGF induces IL-6 synthesis in osteoblasts. In a previous study [12], we have reported that bFGF stimulates phosphoinositide hydrolysis by PLC in MC3T3-E1 cells. Phosphoinositide hydrolysis is generally recognized to result in the formation of inositol 1,4,5-trisphosphates, which mobilize Ca21 from intracellular Ca 21 stores [15]. So, it is probable that bFGF mobilizes Ca21 from intracellular Ca21 stores via PLC activation in MC3T3-E1 cells. To clarify the mechanism of bFGF-induced IL-6 synthesis in these cells, we examined the involvement of Ca21 mobilization from the intracellular Ca 21 stores in the mechanism of bFGF-induced IL-6 synthesis. We here showed that TMB-8, known to inhibit intracellular Ca21 mobilization from intracellular Ca21 stores [27, 28], suppressed the IL-6 synthesis by bFGF. In addition, thapsigargin, which is known to induce Ca21 release from intracellular stores [29], alone induced IL-6 synthesis in MC3T3-E1 cells. So, our findings suggest that Ca21 mobilization from intracellular Ca21 pools is involved in the mechanism of bFGF-induced IL-6 synthesis in osteoblastlike MC3T3-E1 cells. To clarify the role of Ca21 mobilization from extracellular space in the bFGF-induced IL-6 synthesis in MC3T3-E1 cells, we next examined the depletion of extracellular Ca21 by EGTA on the IL-6 synthesis. The bFGF-induced IL-6 synthesis was significantly reduced by chelating extracellular Ca 21 by EGTA. Thus, it seems that intracellular Ca 21 mobilization from extracellular space is involved in the bFGF-induced IL-6 synthesis in MC3T3-E1

cells. In addition, we showed that A23187, a Ca-ionophore, stimulated IL-6 synthesis in these cells. These findings suggest that Ca21 mobilization from extracellular space has a

FIGURE 4. Effect of genistein on bFGF-induced IL-6 synthesis

in MC3T3-E1 cells. The cultured cells were pretreated with various doses of genistein for 20 min, and then stimulated by 30 ng/ ml of bFGF (d) or vehicle (s) with 5 mCi 45Ca21 for 20 min. Each value represents the mean 6 S. D. of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *P , 0.05 vs. control value.

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TABLE 2. Effect of staurosporine or calphostin C on bFGFinduced IL-6 synthesis in MC3T3-E1 cells

TABLE 4. Effect of U-73122 or propranolol on bFGFinduced IL-6 synthesis in MC3T3-E1 cells

IL-6 (pg/ml)

IL-6 (pg/ml)

Control Staurosporine Calphostin C bFGF bFGF 1 staurosporine bFGF 1 calphostin C

14 18 16 168 788 480

6 6 6 6 6 6

3 4 3 14a 72b 35b

The cultured cells were pretreated with 10 nM of staurosporine, 0.1 mM of calphostin C or vehicle for 20 min. The cells were then stimulated by 30 ng/ml of bFGF or vehicle for 48 h. Each value represents the mean 6 S.D. of triplicate determinations. Similar results were obtained with two additional and different cell preparations. aP , 0.05 vs. control value. bP , 0.05 vs. value of bFGF alone.

stimulative effect on IL-6 synthesis in MC3T3-E1 cells. We demonstrated that bFGF truly induced Ca21 influx from extracellular space, even in the presence of nifedipine in MC3T3-E1 cells. In addition, genistein, a protein tyrosine kinase inhibitor [31], suppressed the bFGF-induced Ca21 influx. So, our results suggest that bFGF induces Ca 21 influx from extracellular space through tyrosine kinase activation in MC3T3-E1 cells, and that the Ca21 influx takes part in the mechanism of IL-6 synthesis by bFGF. Taking these findings into account, it is most likely that bFGF stimulates IL-6 synthesis via Ca21 mobilization from both intracellular Ca21 stores and extracellular Ca 21 space in osteoblastlike MC3T3-E1 cells. In a previous study [12], we have reported that bFGF stimulates phosphatidylcholine hydrolysis by PLD independently of phosphoinositide hydrolysis by PLC in osteoblastlike MC3T3-E1 cells, and that the activation of tyrosine kinase is involved in the bFGF-induced phosphatidylcholine hydrolysis. It is well-recognized that phosphoinositide hydrolysis by PLC results in the formation of diacylglycerol, a physiological PKC activator [15, 16]. In addition, it is wellknown that PLD catalyzes phosphatidylcholine hydrolysis, resulting in the formation of phosphatidic acid and choline, and that phosphatidic acid is subsequently degraded to diacylglycerol by phosphatidic acid phosphohydrolase [37, 38]. Therefore, it is probable that bFGF activates PKC through both PLC and PLD in osteoblastlike MC3T3-E1 cells. So, we next examined the role of PKC in the mechanism of TABLE 3. Effect of long-term pretreatment with TPA on

bFGF-induced IL-6 synthesis in MC3T3-E1 cells TPA-pretreatment 2 2 1 1

bFGF 2 1 2 1

IL-6 (pg/ml) 12 173 14 854

6 6 6 6

3 11a 3 64b

The cultured cells were pretreated with 0.1 mM TPA for 24 h, and then stimulated by 30 ng/ml of bFGF or vehicle for 48 h. Each value represents the mean 6 S.D. of triplicate determinations. Similar results were obtained with two additional and different cell preparations. aP , 0.05 vs. control value. bP , 0.05 vs. value of bFGF alone.

Control U-73122 Propranolol bFGF bFGF 1 U-73122 bFGF 1 propranolol

12 13 14 178 341 326

6 6 6 6 6 6

3 3 2 14a 25b 28b

The cultured cells were pretreated with 10 mM of U-73122, 300 mM of propranolol or vehicle for 20 min. The cells were then stimulated by 30 ng/ml of bFGF or vehicle for 48 h. Each value represents the mean 6 S.D. of triplicate determinations. Similar results were obtained with two additional and different cell preparations. aP , 0.05 vs. control value. bP , 0.05 vs. value of bFGF alone.

bFGF-induced IL-6 synthesis in these cells. We here showed that staurosporine significantly enhanced the bFGF-induced IL-6 synthesis in MC3T3-E1 cells. In addition, calphostin C also enhanced the synthesis by bFGF. It is well-known that both staurosporine and calphostin C are inhibitors of PKC [32, 33]. Thus, it seems that the bFGF-induced IL-6 synthesis is suppressed by PKC activation in MC3T3-E1 cells. We next examined the effect of bFGF in the PKC downregulated cells. It has been reported that 24-h pretreatment of TPA (0.1 mM) downregulates PKC in osteoblastlike MC3T3-E1 cells [34], and we also found that the binding capacity of phorbol 12,13-dibutyrate, a PKC-activating phorbol ester [16], in PKC downregulated MC3T3E1 cells is reduced to approximately 30% of the capacity in intact cells [12]. We here showed that PKC downregulation by long-term pretreatment with TPA significantly amplified the bFGF-induced IL-6 synthesis. Therefore, these results suggest that bFGF regulates IL-6 synthesis due to selfinduced PKC activation in MC3T3-E1 cells. To clarify the involvement of phosphoinositide hydrolysis by PLC in the regulation of bFGF-induced IL-6 synthesis in MC3T3-E1 cells, we examined the effect of U-73122, a PLC inhibitor [35], on the IL-6 synthesis. We showed that U-73122 significantly enhanced the bFGF-induced IL-6 synthesis. So, this finding suggests that PKC activation through phosphoinositide hydrolysis by PLC has an inhibitory effect on bFGF by self-induced IL-6 synthesis in MC3T3-E1 cells. In addition, to clarify the involvement of phosphatidylcholine-hydrolyzing PLD in the regulation of bFGF-induced IL-6 synthesis in MC3T3-E1 cells, we examined the effect of propranolol, an inhibitor for phosphatidic acid phosphohydrolase [36], on the IL-6 synthesis. We demonstrated that propranolol also amplified the bFGF-induced IL-6 synthesis in these cells. So, our finding suggests that PKC activation through phosphatidylcholine hydrolysis caused by PLD activation also has an inhibitory effect on self-induced IL-6 synthesis in MC3T3-E1 cells. Therefore, from our findings as a whole, it is most likely that bFGF-induced PKC activation by both PLC and PLD negatively regulates the self-induced IL-6 synthesis in osteoblastlike MC3T3-E1 cells.

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In conclusion, these results strongly suggest that bFGF stimulates IL-6 synthesis, which depends on intracellular Ca 21 mobilization in osteoblastlike cells, and that the IL-6 synthesis by bFGF is autoregulated due to PKC activation by both PLC and PLD.

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