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Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Studies on the role of interleukin-6 (IL-6) in bone metabolism have been accumulating. However, its effects on osteoblasts are still unclear because the results are conflicting depending on the study models employed. We reasoned that these conflicting data are due to variable expression levels of membrane-bound IL-6 receptors (IL-6Rs). In the present study, we found that IL-6 in combination with soluble IL-6R (sIL-6R) consistently caused a marked elevation of alkaline phosphatase and a decrease in proliferation in the human osteoblastic cell line MG-63, which expressed no detectable membrane-bound IL-6R and failed to respond to IL-6. These effects of IL-6/sIL-6R were blocked by neutralizing antibodies to the IL-6 signal transducer gp130, suggesting an involvement of IL-6 signaling in the elicitation of the effects of IL-6/sIL-6R. Upon stimulation with IL-6/sIL-6R, the gp130, cytoplasmic Janus kinases JAK1 and JAK2 were tyrosine phosphorylated. Moreover, signal transducers and activators of transcription STAT1 and STAT3 were also tyrosine phosphorylated, translocated to the nucleus, and bound to the putative STAT-binding DNA elements. In addition, mitogen-activated protein (MAP) kinase was also activated in response to IL-6/sIL-6R. These data demonstrate that sIL-6R may enhance the responsiveness of MG-63 cells to IL-6. Thus, IL-6 in collaboration with sIL-6R may modulate differentiation and proliferation of osteoblastic cells, presumably by activating two distinct signaling pathways of JAK-STAT and MAP kinase.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

INTERLEUKIN-6 (IL-6) is a pleiotropic cytokine that displays diverse biological effects in a variety of cells.1,2 Evidence that IL-6 plays an important role in bone has been accumulating. IL-6 has been shown to stimulate osteoclast-like cell formation3 and bone resorption4 in vitro. More importantly, IL-6 has been implicated in the pathophysiology of decreased bone mass in estrogen-depleted postmenopausal osteoporosis.5–7 It also has been shown that IL-6 is an autocrine/paracrine factor secreted by osteoclasts in Paget's disease8 and Gorham-Stout disease.9

In contrast to these widely recognized effects of IL-6 on osteoclasts, the roles of IL-6 in the function of osteoblastic cells are still controversial because its effects on the phenotype and growth of osteoblast-like cells are conflicting and dependent on the study models employed.10 Human11–13 and mouse4,14 osteoblast-like cells have been demonstrated to produce IL-6 either constitutively or in response to parathyroid hormone (PTH). Expression of IL-6 receptor (IL-6R) mRNA in human osteoblastic cells has also been reported.15 Nonetheless, Littlewood et al. have shown that human osteoblastic cells and rat osteoblastic osteosarcoma cells ROS17/2.8 do not respond to IL-6,16 whereas Fang and Hohn17 observed that IL-6 stimulates DNA synthesis in UMR-106 rat osteoblastic osteosarcoma cells. The reason for this conflict is unknown. Recent studies, however, have reported that soluble forms of IL-6R (sIL-6R) induces marked effects of IL-6 on the cells that do not express membrane-bound IL-6R18 (for review, see Refs. 1, 2, and 19). It has been demonstrated that sIL-6R enhances IL-6 effects on osteoclast-like cell formation20 and IL-6 signaling activation.18,21 We, therefore, reasoned that these conflicting effects of IL-6 may be due to variable expression levels of membrane-bound IL-6 receptors in the osteoblastic cells examined. To explore this, we first determined the effects of the combination of IL-6 and sIL-6R on the well characterized human osteoblastic cell line, MG-63, compared with those of IL-6 alone. MG-63 cells do not express detectable membrane-bound IL-6R and do not respond to IL-6 but exhibit substantial alkaline phosphatase (ALP) activity,22 allowing us to determine IL-6 effects on their differentiation in the absence or presence of sIL-6R.

We next studied the cytoplasmic signaling pathways that were activated in IL-6/sIL-6R-treated MG-63 cells. The signaling of IL-6 is initiated after IL-6 binding to its cell surface receptors, forming a complex of IL-6/IL-6R, followed by the binding of this complex to the membrane-anchored IL-6 signal transducer, gp130,23 which then homodimerizes.18 As downstream of gp130, we focused on the cytoplasmic signaling pathway of the Janus kinases (JAKs) and signal transducers and activators of transcription (STATs), which are known to be involved in IL-6 signaling,1,2 and the Ras-mitogen-activated protein (MAP) kinase pathway, which may play a role in IL-6 signaling.2,24,25 In addition, we attempted to determine the relationship between the activation of these IL-6 signaling pathways and the biological effects of IL-6 in MG-63 cells.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Reagents

Human recombinant IL-6 was purchased from R&D Systems (Minneapolis, MN, U.S.A.). Human recombinant sIL-6R and anti-human gp130 antibodies were described previously.26,27 The antihuman gp130 antibodies were raised against purified recombinant human soluble gp130 and shown to block IL-6–mediated biological responses.27 These antibodies specifically recognized human gp130 and did not cross-react with murine gp130.27 Polyclonal rabbit anti-phosphotyrosine (pTyr) antibodies used in the present study were previously described.28,29 Anti-human JAK1, JAK2, STAT1, STAT3, and MAP kinase (ERK1 and ERK2) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, U.S.A.). In some experiments, mouse monoclonal anti-JAK1, JAK2, and anti-pTyr antibodies purchased from Transduction Laboratories (Lexington, KY, U.S.A.) were also used. These antibodies were raised against synthetic peptides designed based on the specific sequences for each protein and proven to have no cross-reactivity with related family members of proteins.

Cell

The human osteoblastic osteosarcoma cell line MG-63 has been maintained and extensively used in our laboratory.22 These cells were cultured in alpha-modified Eagle's medium (α-MEM; Hazleton Biologics, Inc., Lenexa, KS, U.S.A.) containing 10% fetal bovine serum (FBS; Hyclone Laboratories, Inc., Logan, UT, U.S.A.) and 1% penicillin/streptomycin solution (Life Technologies, Grand Island, NY, U.S.A.).

Morphology of cells

MG-63 cells were treated with test agents for 48 h in α-MEM supplemented with 1% FBS and photographed under phase contrast microscopy.

Determination of cell number

MG-63 cells (5 × 104/24-well) were cultured in the absence or presence of test agents in α-MEM supplemented with 1% FBS. Cells were fed with fresh culture medium and agents every 2 days. After 6 days of culture, cells were trypsinized and counted on a hemocytometer.

ALP microassay

ALP activity in MG-63 cells was determined by the methods described previously.22 Briefly, confluent MG-63 cells were trypsinized and replated in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS in 96-well microtitre plates at 5 × 103 cells/well and allowed to adhere for 48 h before the addition of IL-6, sIL-6R, or both. These agents were diluted in DMEM containing 1% FBS in a separate 96-well microtitre plate before transfer to cells. The cells were incubated with the agents for 6 days, at which time the cultures were washed and lysed with 0.05% Triton X-100. Twenty-five percent of each sample was used to quantify ALP activity using p-nitrophenol-phosphate as a substrate.22 Protein content of the cell layer was determined by assaying 20 μl (10% of the total volume) utilizing the Bio-Rad (Richmond, CA, U.S.A.) reagent. Specific activity was calculated using these two parameters.

Immunoprecipitation and immunoblotting

MG-63 cells were grown to confluency and incubated in α-MEM containing 0.2% FBS for 16 h. Cells were then treated with or without human recombinant IL-6 (200 ng/ml) and/or sIL-6R (500 ng/ml) for 10 minutes at 37°C, washed three times with ice-cold phosphate-buffered saline (PBS), and solubilized in lysis buffer as described previously.29 The supernatants of cell lysates were incubated with antibodies for 4 h at 4°C, followed by immunoprecipitation with protein A-Sepharose (Zymed Laboratories Inc., South San Francisco, CA, U.S.A.) or protein G-agarose (Boehringer Mannheim, Indianapolis, IN, U.S.A.). Immunoprecipitates were washed five times with lysis buffer and boiled in sodium dodecyl sulfate (SDS) sample buffer containing 0.5 M beta-mercaptoethanol. The supernatants were subjected to 7.5% SDS-polyacrylamide gel electrophoresis (PAGE), transferred to nitrocellulose membrane, and immunoblotted with anti-pTyr, JAK1, JAK2, STAT1, STAT3, or MAP kinase antibodies according to the manufacturer's instruction. The samples were visualized with horse-radish peroxidase coupled to protein A (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD, U.S.A.) or horse-radish peroxidase (HRP) coupled anti-mouse IgG antibodies (Cappel, Durham, NC, U.S.A.) using ECL detection kits (Amersham Corp., Arlington Heights, IL, U.S.A.). To further confirm the identification of the tyrosine phosphorylated proteins on the gel, the nitrocellulose membranes were incubated in 0.2 M glycine solution (pH 2.3) for 1 h and reblotted with the corresponding antibodies according to the manufacturer's instructions.

Immunofluorescent staining

MG-63 cells were incubated in α-MEM containing 0.2% FBS for 16 h on glass cover slips. Cells were then treated with or without human recombinant IL-6 (200 ng/ml) and sIL-6R (500 ng/ml) for 30 minutes at 37°C, washed three times with ice-cold PBS, and fixed with 3% paraformaldehyde-PBS for 20 minutes. After 10 minutes of incubation with 0.2% Triton X-100/PBS, cells were treated with 1% bovine serum albumin (BSA)/PBS for 2 h for blocking, incubated with anti-STAT1 monoclonal or anti-STAT3 polyclonal antibodies according to manufacturer's instructions in 1% BSA/PBS, then washed six times with PBS and incubated with fluorescence-conjugated anti-mouse or rabbit IgG antibodies (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, U.S.A.). Cells were washed extensively with PBS and observed under an immunofluorescent microscope (Carl Zeiss Inc., Thornwood, NY, U.S.A.).

Electrophoretic mobility shift assay

Nuclear extracts were prepared as previously reported.30 Nuclear extracts (5 μg) were incubated with 2 μg of poly(dI-dC) for 15 minutes and subsequently incubated with radiolabeled double-stranded oligonucleotides of acute-phase response element (APRE)31 or serum-inducible element of the c-fos promoter (SIE)30 in binding buffer (10 mM HEPES [pH 8.0], 50 mM KCI, 1 mM EDTA, 5 mM MgCI2, 10% glycerol, 5 mM DTT, 0.7 mM polymethylsulfonylfluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 200 μM orthovanadate) for 30 minutes. The sequence of APRE and SIE probes are as follows, respectively: 5′-AGC TTC CTT CTG GGA ATT CCT-3′, 5′-GTC GAC ATT TCC CGT AAA TCG TCG A-3′. DNA–protein complexes were separated by 5% polyacrylamide gel in TBE buffer (89 mM Tris [pH 8.0], 89 mM boric acid, and 2 mM EDTA). For supershift assay, the nuclear extracts were preincubated with anti-STAT1 or -STAT3 antibodies for 30 minutes at room temperature and subjected to electrophoretic mobility shift assay (EMSA), as described.30

MAP kinase assay

MG-63 cell lysates were immunoprecipitated with anti-MAP kinase (ERK2) antibodies and incubated with 10 μCi of32P-γ-ATP (New England Nuclear, Danvers, MA, U.S.A.) and 0.5 mg/ml bovine myelin basic protein (MBP; Sigma Chemical Co., St. Louis, MO, U.S.A.) as a substrate in the presence of 10 mM MgCI2 for 20 minutes at 30°C as described.32 The reaction was terminated by the addition of SDS sample buffer, followed by boiling for 5 minutes, and subjected to 12% SDS-PAGE. The phosphorylated MBP was visualized by autoradiography. The radioactivity of32P-labeled phosphorylated MBP was determined in a liquid scintillation counter.

Statistical analysis

All data were analyzed by analysis of variance followed by a paired “t” test. Values shown are mean ± SEM.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Effects of IL-6/sIL-6R and neutralizing antibodies to gp130 on morphology, ALP activity, and growth of MG-63 cells

IL-6R expression was not detected in MG-63 cells by immunostaining and Western blotting using anti-human IL-6R monoclonal antibody (MT18, kindly provided by Dr. T. Hirano)33 (data not shown). Treatment of MG-63 cells with IL-6 (100 ng/ml) alone did not cause any changes in ALP activity (Fig. 1A, left), growth (Fig. 1A, right), and morphology (Fig. 1B, b), probably due to the lack of membrane-bound IL-6R. In contrast, MG-63 cells exhibited a marked morphological change after 48 h of treatment with a combination of 100 ng/ml IL-6 and 100 ng/ml sIL-6R (Fig. 1B, d). This morphological change was reversed by the addition of 100 μg/ml neutralizing antibody to human gp130 (Fig. 1B, e). The antibody has been shown to neutralize specifically human gp130.27 To determine whether this morphological change was associated with the change in phenotype of MG-63 cells, we next determined the effects of IL-6/sIL-6R on ALP activity. Addition of IL-6/sIL-6R significantly increased ALP activity in MG-63 cells (Fig. 1A, left). In contrast, growth of MG-63 cells was inhibited by IL-6/sIL-6R (Fig. 1A, right). These effects were also blocked by the anti-human gp130 antibodies. We did not observe the effect of either sIL-6R or anti-gp130 antibody alone on MG-63 cells (Figs. 1A, left and right, and 1B, c and f).

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Figure FIG. 1. Effects of IL-6/sIL-6R on ALP activity (A, left), proliferation (A, right) and morphology (B) of MG-63 cells. (A) MG-63 cells (5 × 104/well, 24-well for proliferation assay and 5 × 103/well, 96-well for ALP assay) were treated with none, IL-6 (100 ng/ml), sIL-6R (100 ng/ml), IL-6/sIL-6R, IL-6/sIL-6R with anti-gp130 neutralizing monoclonal antibody (100 μg/ml), or anti-gp130 neutralizing monoclonal antibody for 6 days. ALP activity and cell number were determined as described in the Materials and Methods. IL-6 alone or sIL-6 alone has no effect on proliferation and ALP activity of MG-63 cells. Values are mean ± SEM (n = 4). *Significantly different from control (p < 0.005). **Significantly different from IL-6/sIL-6R (p < 0.005). (B) MG-63 cells were treated with none (a), IL-6 (100 ng/ml) (b), sIL-6R (100 ng/ml) (c), IL-6/sIL-6R (d), IL-6/sIL-6R with anti-gp130 neutralizing monoclonal antibody (100 μg/ml) (e), or anti-gp130 neutralizing monoclonal antibody (f) for 48 h. (Phase contrast microscopy, ×40.) IL-6 alone or sIL-6 alone has no effect on morphological change of MG-63 cells.

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Effects of IL-6/sIL-6R on tyrosine phosphorylation of gp130 in MG-63 cells

IL-6/sIL-6R induced tyrosine phosphorylation of gp130 in MG-63 cells (Fig. 2, left), whereas IL-6 alone showed no effects. Likewise, sIL-6R alone also failed to activate gp130. We confirmed that the tyrosine phosphorylated protein on the gel was the gp130 by reblotting the same membranes with the anti-gp130 antibodies (Fig. 2, right). In other experiments in which we determined dose-dependent effects of IL-6/sIL-6R, we found that 10 minutes of treatment with 200 ng/ml IL-6 and 500 ng/ml sIL-6R of MG-63 cells caused the most profound activation of gp130 (data not shown).

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Figure FIG. 2. Tyrosine phosphorylation of gp130 in MG-63 cells treated with IL-6/sIL-6R. Confluent MG-63 cells were serum starved for 16 h and then stimulated with IL-6 (200 ng/ml)/sIL-6R (500 ng/ml) for 10 minutes, lysed, immunoprecipitated with anti-gp130 antibodies, and immunoblotted with anti-pTyr (left) as described in the Materials and Methods. The same membrane was then reblotted with anti-gp130 antibodies (right).

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Effects of IL-6/sIL-6R on tyrosine phosphorylation of JAKs and STATs in MG-63 cells

JAK1 and JAK2 were tyrosine phosphorylated upon stimulation with IL-6/sIL-6R (Fig. 3A). STAT1 and STAT3 were also tyrosine phosphorylated in response to IL-6/sIL-6R stimulation (Fig. 3B). These tyrosine phosphorylated proteins were identified to be JAK1 and JAK2 (Fig. 3A) and STAT1 and STAT3 (Fig. 3B) after reblotting the same membranes with the corresponding antibodies. The levels of tyrosine phosphorylation of STAT1 or STAT3 reached maximum 10 minutes after the treatment with IL-6/sIL-6R (Fig. 3C).

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Figure FIG. 3. Tyrosine phosphorylation of JAK1 and JAK2 (A), and STAT1 and STAT3 (B and C) in MG-63 cells treated with IL-6/sIL-6R. (A and B) Confluent MG-63 cells were serum starved for 16 h and then stimulated with IL-6 (200 ng/ml)/sIL-6R (500 ng/ml) for 10 minutes, lysed, immunoprecipitated with monoclonal anti-JAK1, polyclonal anti-JAK2, monoclonal anti-STAT1, or polyclonal anti-STAT3 antibodies, and immunoblotted with polyclonal anti-pTyr antibody as described in the Materials and Methods. The same membranes were then reblotted with anti-JAK1, -JAK2, -STAT1, or -STAT3 antibodies, respectively. (There is no heavy chain for anti-JAK1 and STAT1 followed by immunoblotting using polyclonal anti-pTyr antibodies, because the primary antibodies were mouse monoclonal antibodies which are not visualized by HRP-protein A.) (C) Confluent MG-63 cells were serum starved for 16 h and then stimulated with IL-6 (200 ng/ml)/sIL-6R (500 ng/ml) for 30 s, 3, 5, 10, 30 minutes, lysed, immunoprecipitated with monoclonal anti-STAT1, or polyclonal anti-STAT3 antibodies, and immunoblotted with polyclonal anti-pTyr antibody as described in the Materials and Methods.

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Effects of IL-6/sIL-6R on DNA binding of STATs

Using the immunofluorescent staining technique, STAT1 and STAT3 were found to translocate and concentrate in the nucleus 30 minutes after stimulation with IL-6/sIL-6R (Fig. 4).

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Figure FIG. 4. Translocation and concentration of STAT1 (a, c) and STAT3 (b, d) in the nucleus in response to IL-6/sIL-6R stimulation. Unstimulated (a, b); stimulated with IL-6 (200 ng/ml)/sIL-6R (500 ng/ml) for 30 minutes (c, d). Immunofluorescent staining was performed with anti-STAT1 (a, c) or anti-STAT3 (b, d) antibodies as described in the Materials and Methods. Note diffuse distribution of STAT proteins in the cytoplasm of unstimulated cells (a, b) and concentration of STAT proteins in the nuclei of stimulated cells (b, d).

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To examine the DNA binding activity of activated STATs, binding of nuclear extracts of MG-63 cells treated with or without IL-6/sIL-6R with radiolabeled oligonucleotides of APRE and SIE was determined by EMSA. APRE1,31 and SIE30,34 are well known to form complexes with activated STATs. APRE (Fig. 5A, lane 3) and SIE (Fig. 5A, lane 7) probes associated with the nuclear extracts of MG-63 cells stimulated with IL-6 and sIL-6R. The bindings were specifically blocked by 50 times excess amounts of corresponding cold probes (Fig. 5A, lanes 4 and 8). Moreover, preincubation of the MG-63 cell nuclear extracts with anti-STAT1 and -STAT3 antibodies caused a supershift of the APRE- and SIE-binding complex (Fig. 5B, lanes 4 and 8 and Fig. 5C, lanes 4 and 8, respectively). These results showed that STAT1 and STAT3 were activated, translocated into the MG-63 cell nucleus, and bound with APRE and SIE upon stimulation with IL-6/sIL-6R. These data also indicate that both STAT1 and STAT3 bind to APRE and SIE to form a heterotrimer complex after IL-6/sIL-6R stimulation.

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Figure FIG. 5. EMSA using the nuclear extracts of MG-63 cells (A) and super-shift in the presence of anti-STAT1 (B) and -STAT3 (C) antibodies. (A) MG-63 cells were serum starved for 16 h and then stimulated with or without IL-6 (200 ng/ml)/sIL-6R (500 ng/ml) for 20 minutes. Nuclear extracts of unstimulated (−) (lanes 2 and 6) and stimulated (+) (lanes 3, 4, 7, and 8) MG-63 cells were subjected to EMSA using32P-labeled oligonucleotides of APRE (left) and SIE (right) as probes. Arrows indicate an association of nuclear extracts of stimulated MG-63 cells with APRE (lane 3) or SIE (lane 7). Lanes 1 and 5 were loaded with only radiolabeled probes. Fifty-fold excess of corresponding unlabeled probes were used for competitive inhibition assay (lanes 4 and 8). (B, C) Nuclear extracts of unstimulated (−) (lanes 1, 3, 5, and 7) and stimulated (+) (lanes 2, 4, 6, and 8) MG-63 cells were preincubated with anti-STAT1 (B, lanes 3, 4, 7, and 8) and -STAT3 (C, lanes 3, 4, 7, and 8) antibodies for 1 h and subjected to EMSA.

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Effects of IL-6/sIL-6R on MAP kinase

IL-6/sIL-6R caused tyrosine phosphorylation of MAP kinase (Fig. 6A, left). Reblotting the same membranes with the anti-MAP kinase antibodies revealed that the tyrosine phosphorylated protein was MAP kinase (Fig. 6A, right), and the mobility of MAP kinase was shifted due to the phosphorylation by IL-6/sIL-6 stimulation. Furthermore, MAP kinase activity determined using MBP as a substrate was increased upon stimulation with IL-6/sIL-6R in a time-dependent manner (Fig. 6B). Activation of MAP kinase was observed 3 minutes after the stimulation and before the initiation of STAT1 and STAT3 activation (Figs. 3C and 6B).

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Figure FIG. 6. Activation of MAP kinase by IL-6/sIL-6R. (A) Tyrosine phosphorylation of MAP kinase. MG-63 cell lysates were immunoprecipitated with anti-MAP kinase antibodies (anti-ERK2 antibodies), subjected to SDS-PAGE, and immunoblotted with anti-pTyr antibodies (left) or anti-MAP kinase antibodies (anti-ERK2 antibodies) (right). We obtained identical results using anti-ERK1 antibodies (data not shown). (B) In vitro MAP kinase assay. MG-63 cells were serum starved for 16 h and then stimulated with IL-6 (200 ng/ml)/sIL-6R (500 ng/ml) for 30 s, 3, 5, 10, and 30 minutes. Cells were lysed, immunoprecipitated with anti-MAP kinase antibodies (anti-ERK2 antibodies), and subjected to MAP kinase assay using bovine MBP as a substrate. Numbers shown at the bottom of each lane represent radioactivity of the MBP bands determined in a scintillation counter after the corresponding bands on the gel were excised.

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Effects of aFGF and bFGF on tyrosine phosphorylation of JAKs, STATs, and MAP kinase in MG-63 cells

It has been shown that aFGF and bFGF stimulate MAP kinase but do not activate JAKs and STATs.35 Consistent with this previous report,35 aFGF and bFGF (100 ng/ml) strongly induced tyrosine phosphorylation of MAP kinase (Fig. 7) but failed to cause tyrosine phosphorylation of JAK1, JAK2, STAT1, and STAT3 (Fig. 7). However, in the same experiment, IL-6/sIL-6R caused tyrosine phosphorylation of JAK1, JAK2, STAT1, STAT3, and MAP kinase (Fig. 7).

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Figure FIG. 7. Tyrosine phosphorylation of JAKs, STATs, and MAP kinase by IL-6/sIL-6R, aFGF, or bFGF stimulation. Confluent MG-63 cells were serum starved for 16 h and then treated with IL-6 (200 ng/ml)/sIL-6R (500 ng/ml), aFGF (100 ng/ml), or bFGF (100 ng/ml) for 10 minutes, lysed, immunoprecipitated with anti-MAP kinase, JAK1, JAK2, STAT1, or STAT3 antibodies, and immunoblotted with anti-pTyr as described in the Materials and Methods.

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Effects of aFGF and bFGF on morphology, ALP activity, and growth of MG-63 cells

aFGF and bFGF (10 ng/ml) did not stimulate ALP activity (Fig. 8, left) and change cell morphology (data not shown) but increased proliferation of MG-63 cells (Fig. 8, right). These effects of FGFs were in contrast to those of IL-6/sIL-6R, which significantly increased ALP activity (Fig. 8, left) and decreased cell proliferation (Fig. 8, right).

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Figure FIG. 8. Effects of IL-6/sIL-6 and aFGF and bFGF on ALP (left) and proliferation (right) of MG-63 cells. Cells (5 × 104/well, 24-well for proliferation assay and 5 × 103/well, 96-well for ALP assay) were treated with 100 ng/ml IL-6/100 ng/ml sIL-6R, 10 ng/ml aFGF, or 10 ng/ml bFGF for 6 days. ALP activity and cell number were determined as described in the Materials and Methods. Values are mean ± SEM (n = 4). *Significantly different from control (p < 0.01).

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

In the present study, using the human osteoblastic cell line MG-63, which does not express detectable membrane-bound IL-6R and fails to respond to IL-6, we have shown that combination of IL-6 and sIL-6R caused morphological change, increase in ALP activity, and decrease in cell proliferation. IL-6 or sIL-6R alone showed no effects on MG-63 cells. Our results, that sIL-6R induces IL-6 effects on MG-63 cells, suggest that the previous conflicting data on IL-6 effects on osteoblastic cells may be the result of variable expression levels of membrane-bound IL-6R among the cells studied.

Since neutralizing monoclonal antibody to gp130 blocked the effects of IL-6/sIL-6R on ALP activity and proliferation, it is probable that gp130 mediates elicitation of the biological effects of IL-6/sIL-6R in MG-63 cells. Recent studies have reported that ciliary neurotrophic factor and leukemia inhibitory factor, which also utilize gp130 as their signal transducer, increase ALP activity in mouse osteoblastic MC3T3-E1 cells.36 These results together with our data suggest that gp130 plays a key role in mediating the effects of IL-6 and its family members of cytokines in osteoblast-like cells. Since supplementation of sIL-6R dramatically caused activation of gp130, it seems likely that cells that express none or little membrane-bound IL-6R are able to respond to IL-6 in the presence of sIL-6R as long as they express gp130. In this regard, it is of note that gp130 is ubiquitously expressed in numerous types of cells,1,2 and that circulating levels of sIL-6R are maintained at substantially high levels in normal individuals (approximately 70 ng/ml).19

We have also reported that plasma levels of sIL-6R in normal mice are between 20 and 30 ng/ml.37 One reason for high circulating levels of sIL-6R might be to spatially and temporally induce biological responses to IL-6 in cells that do not express membrane-bound IL-6R but possess gp130. Alternatively, it is possible that sIL-6R may bind to extra IL-6, which exceeds the concentration of membrane-bound IL-6R on target cells. We speculate that when IL-6 production by osteoblasts is increased due to pathological changes such as inflammation and estrogen depletion in the bone milieu, circulating sIL-6R might assist the membrane-bound IL-6R in the binding to excessive amounts of IL-6, thereby enhancing the response to IL-6.

In parallel with the induction of these biological responses, IL-6/sIL-6R also caused tyrosine phosphorylation of the gp130. Bellido et al.38 also reported that IL-6 induced tyrosine phosphorylation of gp130 in osteoblast-like cells. However, we have further shown that IL-6/sIL-6R also caused activation of the cytoplasmic tyrosine kinases JAK1 and JAK2 and latent cytoplasmic transcription factors STAT1 and STAT3. These activated STATs subsequently translocated and bound to the putative STAT-binding DNA elements in the nucleus. Moreover, we also clearly demonstrated that IL-6/sIL-6R provoked concomitant activation of the MAP kinase pathway. Thus, in the present study, we demonstrated that two distinct cytoplasmic signaling pathways, JAK-STAT and MAP kinase, were activated and involved in nuclear events during IL-6 signaling in osteoblastic MG-63 cells.

The relationship between an elicitation of the biological effects of IL-6 and an activation of these two signaling pathways in MG-63 cells is complex. Our data confirmed that FGFs failed to activate the JAK-STAT pathway but activated the MAP kinase pathway in MG-63 cells. FGFs did not increase the ALP activity of MG-63 cells. In contrast, IL-6/sIL-6R, which activated both JAK-STAT and MAP kinase pathway, increased ALP activity. These data suggest that activation of MAP kinase alone is not sufficient to stimulate ALP activity and that concomitant and harmonious activation of JAK-STAT and MAP kinase pathways is necessary for stimulation of ALP activity in MG-63 cells.

It is not determined in the present study whether these two pathways independently or dependently mediate the effects of IL-6 in MG-63 cells. However, recent studies demonstrate that serine phosphorylation of STAT1 and STAT3 by MAP kinase is necessary for maximal stimulation of gene transcription-regulating activity of STAT1 and STAT3.39,40 It is, therefore, possible that similar cross-talk between JAK-STAT and MAP kinase pathways occurs in MG-63 cells which are stimulated with IL-6/sIL-6R. Indeed, we observed that MAP kinase activation preceded STAT1 and STAT3 activation. Furthermore, our preliminary data show that overexpression of dominant negative Ras abolishes not only MAP kinase activation but also inhibits STAT activation by IL-6/sIL-6R and that an increase in ALP activity induced by IL-6/sIL-6R is also significantly impaired by the dominant-negative Ras (manuscript in preparation).

In conclusion, the present study demonstrates the supplementary role of sIL-6R in the exertion of biological effects of IL-6 in osteoblast-like MG-63 cells. In addition, our results also demonstrate that dual but distinct signaling pathways, JAK-STAT and MAP kinase, are concomitantly activated in MG-63 cells in response to sIL-6R/IL-6. This study should provide a clue to link the activation of these cytoplasmic signaling pathways with the elicitation of the biological responses to IL-6 in the osteoblastic cells.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

The authors thank Dr. Toshio Hirano, Division of Molecular Oncology, Biomedical Research Center, Osaka University Medical School for providing anti-human IL-6R monoclonal antibodies and Thelma Barrios and Nancy Garrett for their excellent secretarial assistance in preparing the manuscript. This work was supported by National Institutes of Health grants PO1-CA40035, PO1-AR39529, R37-AR28149, and RO1-DK45229.

References

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References
  • 1
    Kishimoto T, Taga T, Akira S 1994 Cytokine signal transduction Cell 76:253262.
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