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Endocrinology Vol. 138, No. 9 3666-3676
Copyright © 1997 by The Endocrine Society

ARTICLES

Activation of the Janus Kinase/STAT (Signal Transducer and Activator of Transcription) Signal Transduction Pathway by Interleukin-6-Type Cytokines Promotes Osteoblast Differentiation1

Teresita Bellido, Victoria Z. C. Borba, Paula Roberson and Stavros C. Manolagas

Division of Endocrinology and Metabolism, Center for Osteoporosis and Metabolic Bone Diseases, University of Arkansas for Medical Sciences, and the McClellan Veterans Administration Medical Center, Geriatric Research, Education, and Clinical Center, Little Rock, Arkansas 72205

Address all correspondence and requests for reprints to: Teresita Bellido, Ph.D., Division of Endocrinology and Metabolism, University of Arkansas for Medical Sciences, 4301 West Markham, Mail Slot 587, Little Rock, Arkansas 72205. E-mail: TMBELLIDO{at}LIFE.UAMS.EDU


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have previously established that stromal/osteoblastic cells collectively express receptors for all members of the cytokine subfamily that share the gp130 signal transducer and that different receptor repertoires may be expressed at different stages of differentiation of this lineage. We have now used human (MG-63) and murine (MC3T3-E1) osteoblastic cell lines as well as primary murine calvaria cells to test the hypothesis that these receptors mediate effects of the cytokines on the biology of osteoblasts. We report that as in other cell types, all of the osteoblastic cell models responded to interleukin-6 (IL-6)-type cytokines with activation of both the JAK/STAT (Janus kinase/signal transducer and activator of transcription) and the mitogen-activated protein kinase (MAPK) pathways. In addition, IL-6-type cytokines stimulated alkaline phosphatase activity and osteocalcin expression and inhibited (MG-63), stimulated (MC3T3-E1), or had no effect (calvaria cells) on the rate of cell proliferation. The ability of a given cell type to respond to a particular member of this family of cytokines was strictly dependent on the presence of the corresponding ligand-binding subunit ({alpha}) of the cytokine receptor, and the magnitude of all the effects was closely correlated with the concentration of this subunit. The relative contribution of the JAK/STAT and MAPK pathways to the biological effects of the cytokines was evaluated using kinase inhibitors. Cytokine-mediated modulation of cell proliferation as well as stimulation of alkaline phosphatase activity were abrogated by tyrosine kinase inhibitors as well as a threonine/serine kinase inhibitor, but were only minimally affected by a specific inhibitor of MAPK phosphorylation. These results demonstrate that IL-6-type cytokines, besides their osteoclastogenic properties, promote differentiation of committed osteoblastic cells toward a more mature phenotype and that this action is mediated primarily via the activation of the JAK/STAT pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INTERLEUKIN-6 (IL-6), IL-11, leukemia inhibitory factor (LIF), ciliary neurotropic factor (CNTF), oncostatin M (OSM), and cardiotropin-1 comprise a group of cytokines with overlapping effects in many cell types (1, 2). Such redundancy is probably due to the fact that they share receptor components and signal transduction pathways (3, 4). This group of cytokines is referred to hereafter as IL-6-type cytokines (2).

The general mechanism of action of the IL-6-type cytokines has been well established. According to this mechanism, binding of the cytokines to the ligand-recognizing component of the receptor ({alpha}-subunit) leads to the formation of homo- or heterodimers of gp130, a shared signal-transducing component (ß-subunit) for all of these receptors. gp130 homodimers are used by IL-6 and IL-11, whereas heterodimers of gp130 with an additional ß-subunit are used by LIF, CNTF, OSM, and cardiotropin-1. Cytokine-induced dimerization of the ß-subunits activates kinases of the Janus family (JAKs) and induces the recruitment, tyrosine phosphorylation, and nuclear translocation of signal transducers and activators of transcription (STATs) (1, 3, 5, 6). Besides the JAK/STAT pathway, mitogen-activated protein kinases (MAPKs), also called extracellular signal-regulated protein kinases (Erks), might be involved in the membrane to nucleus signaling by IL-6-type cytokines. Thus, IL-6-type cytokines are known to increase tyrosine phosphorylation of MAPKs in several cell types (7, 8), and phosphorylation of a potential site of MAPK action in the C-terminus of STATs is required for maximal transcriptional activation (6).

Extensive evidence accumulated during the last few years has established that IL-6-type cytokines play a profound role in bone metabolism. Thus, IL-6 and IL-11 are produced in nanomolar concentrations by cells of the stromal/osteoblastic lineage in response to stimulation by PTH, PTH-related peptide, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], transforming growth factor-ß, platelet-derived growth factor, as well as IL-1 and tumor necrosis factor (9). Acting via stromal/osteoblastic cells, IL-6, IL-11, OSM, and LIF are potent stimulators of the development of osteoclasts from their hematopoietic progenitors (10). Consistent with its osteoclastogenic property, increased production of IL-6 has been implicated in the pathophysiology of many different disease states characterized by increased bone resorption, including postmenopausal osteoporosis, Paget’s disease, hyperparathyroidism, multiple myeloma, hyperthyroidism, rheumatoid arthritis, McCune-Albright syndrome, and Gorham-Stout disease (11). The osteoclastogenic effects of IL-6 in vitro can only be demonstrated when exogenous soluble IL-6 receptor (sIL-6R) is provided (10), indicating that expression of the {alpha}-subunit of the IL-6R in bone is a limiting factor for the effects of the latter cytokine (12). In agreement with the idea that IL-6 attains its importance for bone metabolism in pathological states where there is increased IL-6 production in combination with increased sensitivity to the effects of IL-6, an increase in IL-6R production has been also noted in the above-listed disease states (11, 13).

We have previously established that cells of the stromal/osteoblastic lineage collectively express all of the {alpha}- and ß-subunits of the receptors for IL-6, IL-11, LIF, CNTF, and OSM. However, different repertoires of {alpha}- and ß-subunits were present in cells with distinct phenotypes, suggesting that the expression of these receptors is a function of the stage of cell differentiation (12). Based on evidence that besides their osteoclastogenic properties, IL-6-type cytokines may also affect bone formation (11), in the studies of the present report we have employed established osteoblast-like cell lines of human and murine origin as well as primary cultures of murine calvaria cells and have searched for direct effects of IL-6-type cytokines on the biology of osteoblasts. In addition, we have attempted to dissect the role of the JAK/STAT from the role of the MAPK signaling pathways in the mediation of the cytokine action on these cells. Our results demonstrate that IL-6-type cytokines exert direct effects on cells of the osteoblastic lineage in a receptor ({alpha}-subunit)-restricted manner. Moreover, they suggest that besides their osteoclastogenic properties, IL-6-type cytokines can promote differentiation of committed osteoblastic cells toward a more mature phenotype, and this action is mediated primarily via the activation of the JAK/STAT pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Human IL-6 and LIF were obtained from Upstate Biotechnology (Lake Placid, NY). IL-11 was purchased from Genzyme (Cambridge, MA). sIL-6R and recombinant human OSM were obtained from R&D Systems (Minneapolis, MN). CNTF and soluble CNTF receptor were provided by Dr. Neil Stahl (Regeneron Pharmaceuticals, Tarrytown, NY). 1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] was provided by Dr. Milan Uskokovic (Hoffman LaRoche, Nutley, NJ). FBS, nonimmune IgG, myelin basic protein (MBP), phorbol 12-myristate 13-acetate (PMA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), NAD, L (+) lactic acid, diaphorase, alkaline phosphatase (AP) buffer, p-nitrophenyl phosphate (Sigma 104), ribonuclease A, and propidium iodide were purchased from Sigma Chemical Co. (St. Louis, MO). MEM and RPMI 1640 medium were obtained from Life Technologies (Grand Island, NY). 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS), was purchased from Promega (Madison, WI). Mouse monoclonal anti-STAT1{alpha} antibody (sc-417) and rabbit polyclonal anti-STAT3 antibody (sc-482) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Cell culture conditions
Murine calvaria cells were obtained from neonatal (3- to 6-day-old) Swiss-Webster mice as previously described (14). Briefly, calvaria were incubated in 4 mM EDTA in PBS at 37 C for three 10-min periods; supernatants were discarded. Subsequently, calvaria were rinsed in PBS and subjected to digestion with 200 U/ml collagenase in PBS for four 15-min periods. Cells released during the first digestion were discarded. Cells released during the subsequent digestions were combined and cultured in RPMI 1640 supplemented with 5% FBS (1–2 x 10-4 cells/cm2). The murine calvaria-derived osteoblast-like cell line MC3T3-E1 (15) and the human osteosarcoma cell line MG-63 (16) were cultured in phenol red-free MEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cultures were kept in a humidified atmosphere of 5% CO2 in air at 37 C. The cytokine receptor repertoire of these cells is summarized in Table 1Go. MG-63 cells express limited amounts of receptors for IL-6 and IL-11, greater amounts of type II receptors for OSM, and no receptors for LIF. MC3T3-E1 cells and murine calvaria cells both express receptors for LIF, CNTF, and IL-11, but do not express functional receptors for IL-6.


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  		Table 1. Summary of expression of receptor components for IL-6-type cytokines in the osteoblast-like cells used in this study

 
Thymidine incorporation and cell number measurements
The rate of [3H]thymidine incorporation was determined as described previously (17). Cells were plated in 10% FBS-containing medium at a density of 0.75–0.9 x 104 cells/cm2 in 24-well plates and cultured for 24 h. Subsequently, media were replaced with fresh media containing 2% FBS. Cells (four replicas per condition) were cultured for 3 days in the absence or presence of the indicated cytokines. Subsequently, [3H]thymidine (0.5 µCi/well) was added 16 h before harvesting the cells. Intact DNA and associated [3H]thymidine were precipitated in ice-cold 5% trichloroacetic acid, and the radioactivity was counted. Cell number was estimated by measuring the reduction of MTT to the formazan product, as previously described (18). Twenty-five microliters of 5 mg/ml MTT were added, and cells were incubated for 2 h at 37 C. Subsequently, 100 µl 20% SDS in 50% dimethylformamide were added, and after 16-h incubation at 37 C, OD was measured at 570 nm in an enzyme-linked immunosorbent assay plate reader. The absolute cell number was determined by plotting the OD value of the unknown sample in a standard curve of OD values obtained by using a range of known number (counted) of cells for each type.

Cell viability and in situ end labeling of fragmented DNA
The percentage of viable cells in control or cytokine-treated cultures was evaluated by dye exclusion. Cells were released from the culture dish using trypsin-EDTA combined with 0.4% trypan blue and counted using a hemocytometer. A minimum of 400 cells were counted. Cells exhibiting nuclear and cytoplasmic trypan blue staining were considered dead. In situ end labeling of fragmented DNA was assessed as previously described (19, 20). Briefly, MG-63 cells were plated in 8-well chamber slides at 1.5 x 104 cells/cm2 and cultured for 24 h in 10% FBS-containing medium. Subsequently, cells were treated with 1 or 10 ng/ml OSM, IL-6, or IL-11 in 2% FBS-containing medium for 1–4 days. Cell monolayers were washed with PBS; fixed in methanol; rinsed in a buffer containing 50 mM Tris-HCl, 5 mM MgCl2, 10 mM 2-mercaptoethanol, and 0.005% BSA, pH 7.5; and air-dried. Slides were than incubated for 90 min at 15 C with the same buffer containing 0.01 mM deoxy-ATP, deoxy-CTP, deoxy-GTP, and biotin-11-deoxy-UTP in the presence of 20 U/ml Klenow fragment of DNA polymerase I. Nucleotide incorporation was visualized using avidin-conjugated horseradish peroxidase.

Cell cycle analysis
MG-63 cells were plated in 6-well plates in medium containing 10% FBS at a density of 0.25 x 106 cells/well and cultured for 24 h. Subsequently, media were removed and replaced with fresh media containing 5% FBS, and cells were cultured in the absence or presence of OSM, LIF, IL-11, IL-6, or IL-6 in combination with sIL-6R. After 24 h, cells were harvested by trypsinization, washed with PBS, and fixed overnight with 70% ethanol in PBS at 4 C. Before staining, cells were washed three times with PBS and resuspended in PBS containing 150 U/ml ribonuclease A and 5 pg/ml propidium iodide (21). Flow cytometric analysis was performed using a FACScan (Becton Dickinson, San Jose, CA). Data were analyzed using Cellfit, and DNA histograms were plotted with Lysis software (Becton Dickinson). The distribution of cells in the different phases of the cell cycle was expressed as a percentage of the total number of cells. Approximately 10,000 cells/sample were analyzed.

AP activity
Cells were plated in the appropriate medium containing 10% FBS and cultured to 85–90% confluence (36–48 h). Media were removed and replaced with fresh media containing 5% FBS, and cells (four replicas per condition) were cultured in the absence or presence of 10 ng/ml of the cytokines alone or in combination with 1 µg/ml of the respective soluble receptors (for IL-6 and CNTF) or 1,25-(OH)2D3, as indicated. After 4 days of treatment, cell monolayers were washed with PBS and incubated for 1 h at 4 C in a buffer containing 100 mM glycine, 1 mM MgCl2, and 1% Triton X-100, pH 10.5. AP activity in the lysates was assayed using p-nitrophenyl phosphate (Sigma 104) as a substrate, and the production of p-nitrophenol was determined by measuring the absorbance at 410 nm (22). The protein concentration was assayed by the method of Bradford using BSA as a standard (23). AP activity was expressed as units per mg protein/min (1 U = 10 µg p-nitrophenol). For the experiments using kinase inhibitors, MG-63 cells were suspended in medium containing 10% FBS and cultured in 96-well plates to 85–90% confluence. Medium was removed and replaced with fresh medium containing 5% FBS and the indicated concentrations of the kinase inhibitors. After 30 min of pretreatment with the inhibitors, OSM (10 ng/ml) was added to the appropriate wells. Cells (eight replicas per condition) were cultured for 3 days. Media were removed, and cells were fixed in 10% formalin PBS buffer. The amount of cells per well was estimated using an assay based on the reduction of the tetrazolium derivative, MTS, coupled to lactate dehydroxylase (LDH), with a microtiter plate reader. Briefly, monolayers were washed once with distilled water and three times with 0.2 M Tris, pH 8.2, and then 75 µl 0.2 M Tris, pH 8.2, were added to each well. Subsequently, 75 µl 0.2 M Tris, pH 8.2, containing 1.3 x 10-3 M NAD, 5.4 x 10-2 M L (+) lactic acid, 6 mg/ml MTS, and 2.572 U/ml diaphorase were added to each well, and readings at 490 and 750 nm were taken at 0, 5, 10, 20, 30, 45, 60, 90, and 120 min in a plate reader using Time Management software (Dynatech Laboratories, Chantilly, VA). After MTS/LDH detection, cells were washed three times with 20 mM HEPES, 150 mM NaCl, and 1 mM MgCl2, pH 7.4. Subsequently, 75 µl AP buffer and 75 µl AP substrate were added to each well, and readings at 410 and 750 nm were taken at the same time points. Results are expressed as ratios of AP rate (OD per min) to MTS-LDH rate (OD per min).

RNA extraction and Northern blot analysis
Total cellular RNA was isolated from 85–90% confluent cell cultures treated with cytokines for 24 h, and polyadenylated RNA was selected as previously described (12). Messenger RNA (mRNA) was separated by electrophoresis in 1% agarose formaldehyde gels, transferred to nylon membranes, and fixed by heating at 80 C under vacuum for 2 h. Blots were probed with radiolabeled complementary DNAs for rat osteocalcin (provided by Drs. J. Lian and G. Stein) (24), for AP (obtained from American Type Culture Collection, Rockville, MD), or for the housekeeping gene Cho-B (12, 25) and analyzed using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Preparation of nuclear extracts and electrophoretic mobility shift assays (EMSA)
MG-63 cells were cultured until 85–90% confluence and were maintained in serum-free medium for 2 h before stimulation. Cytokines alone (50 ng/ml) or IL-6 in combination with sIL-6R (1 µg/ml) were added to cell monolayers and maintained for 15 min at 37 C. Cells were rinsed once with PBS, and nuclear extracts were prepared as previously described (26). Briefly, 4–7 x 107 cells were washed with hypotonic buffer [10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, and 0.5 mM dithiothreitol (DTT)] and lysed for 10 min on ice in 30 µl/107 cells of the same buffer containing 0.1% Nonidet P-40. Lysates were centrifuged at 10,000 x g at 4 C for 10 min. Pelleted nuclei were resuspended in 30 µl/107 cells of lysis buffer [20 mM HEPES (pH 7.9), 420 mM NaCl, 1 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, and 25% glycerol] and were incubated at 4 C for 15 min. Lysed nuclei were dispersed and centrifuged at 10,000 x g at 4 C for 10 min. Supernatants were collected, snap-frozen, and stored at -70 C. Protein concentrations in the nuclear extracts were determined by the method of Bradford (23). A synthetic, double stranded oligonucleotide containing a palindromic sequence element (indicated in boldface) that binds to STAT complexes (5'-CTAGTGCTTCCCGGAACGT-3') (27) was end labeled using [{gamma}-32P]ATP and T4 polynucleotide kinase. End-labeled probe (1 ng) was incubated for 20 min at room temperature with 10 µg nuclear proteins in a solution containing 50 µg/ml double stranded salmon sperm DNA, 6% glycerol, 10 mM HEPES (pH 7.5), 80 mM KCl, 1 mM EDTA, and 1 mM EGTA in the absence or presence of a 100-fold molar excess of unlabeled oligonucleotide. Supershift experiments were performed by incubating the nuclear proteins with either nonimmune IgG, anti-STAT1{alpha} antibody or anti-STAT3 antibody for 10 min at room temperature before addition of the labeled probe.

Kinase assays in MBP-containing gels
Confluent MG-63 cell cultures were maintained for 16 h in medium without serum containing 1% BSA and were subsequently stimulated with the cytokines alone (50 ng/ml), IL-6 in combination with sIL-6R (1 µg/ml), or 10-7 M PMA for 15 min unless indicated otherwise. Monolayers were washed twice with cold PBS and lysed in a buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 10 mM NaF, 1 mM sodium orthovanadate, 5 µg/ml leupeptin, 0.14 U/ml aprotinin, 1 mM phenylmethylsulfonylfluoride, and 1% Triton X-100. Cells were scraped off the plates, incubated on ice for 10 min, and centrifuged for 15 min at 14,000 x g. The protein concentration was measured using a Bio-Rad detergent compatible kit (Hercules, CA). MAPK activity in the cell lysates was measured using an in-gel kinase assay previously described (28). Briefly, protein extracts (50 µg/lane) were dissolved in buffer for protein electrophoresis and separated on SDS-polyacrylamide (7.5%) gels containing 0.5 mg/ml MBP. Proteins were fixed by incubating the gel for 2 h at room temperature with 20% 2-propanol in 50 mM Tris-HCl, pH 8.0, and 5 mM 2-mercaptoethanol (TME). SDS was removed from the gel by washing with TME for 1 h at room temperature. The enzyme was denatured by treating the gel with 6 M guanidine HCl in TME. Subsequently, the enzyme was renatured by incubating the gel with 0.04% Tween-40 in TME. After renaturation, the gel was preincubated at 25 C for 1 h with 40 mM HEPES, pH 8.0, containing 2 mM DTT and 10 mM MgCl2. Phosphorylation of MBP was carried out by incubating the gel at 25 C for 1 h with 40 mM HEPES (pH 8.0), 2 mM DTT, 0.5 mM EGTA, 10 mM MgCl2, 40 µM ATP, and 25 µCi [{gamma}-32P]ATP. The gel was subsequently washed with a 5% (wt/vol) trichloroacetic acid solution containing 1% sodium pyrophosphate until the radioactivity of the solution became negligible. The gel was then dried and subjected to autoradiography.

Western blot analysis with antiphospho-MAPK and anti-MAPK antibodies
The phosphorylation status of MAPK (Erk1 and Erk2) was analyzed by immunoblotting the cell lysates obtained above with an antibody that recognizes tyrosine-phosphorylated MAPK (phospho-MAPK antibody) and a control antibody that recognizes phosphorylated and unphosphorylated MAPK (MAPK antibody; New England Biolabs, Berverly, MA). Proteins (100 µg/lane) were run on 7.5% SDS-polyacrylamide gels and electrophoretically transferred to polyvinylidene difluoride (New England Nuclear, Boston, MA). Membranes were blocked for 1 h at room temperature in PBS containing 0.1% Tween-20 and 5% nonfat dry milk, and subsequently incubated overnight at 4 C with a 1:1000 dilution of either phospho-MAPK or MAPK antibody, followed by incubation for 1 h with the secondary antirabbit antibody conjugated with AP. Blots were developed using an AP-based chemiluminescence assay, according to the manufacturer’s recommendations (New England Biolabs).

Statistical analysis
Data were analyzed by one-way ANOVA, and Dunnett’s test was used to estimate the level of significance of differences between means. For statistical analysis of the data regarding kinase inhibitor effects on OSM-mediated responses (Fig. 7Go), standardized OSM AP/MTS values were calculated by dividing each replica of the OSM-treated group by the mean of the corresponding basal group. These values were analyzed by one-way ANOVA, and subsequently, each inhibitor group was compared with the group without inhibitor using Dunnett’s test and an experimentwise significance level of 0.05. Because PD98059 specifically inhibits the activity of MEK (the enzyme responsible for MAPK activation), whereas all the other agents used inhibit either tyrosine or serine/threonine kinases, an analysis using Dunnett’s test to compare all other inhibitor groups to the group treated with PD98059 was also conducted. Data comparing the distributions of cells in the different phases of the cell cycle (Fig. 2Go) were analyzed using the {chi}2 test.



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  		Figure 7. Effects of kinase inhibitors on the OSM-mediated effects on human osteoblastic cells. Semiconfluent cultures of MG-63 cells were pretreated with the indicated kinase inhibitors 30 min before the addition of OSM (10 ng/ml). A, Cells were cultured for 1 day, and the percentage of cells in each phase of the cell cycle was determined, as described in Fig. 2Go. Similar results were obtained in two additional experiments. B, AP activity corrected by the number of cells (AP/MTS) was determined after 3 days of culture in the presence of OSM, as detailed in Materials and Methods. Data are expressed as a percentage of the control values and are representative of five experiments performed. The standardized OSM AP/MTS values were analyzed by one-way ANOVA and compared with the group without inhibitor using Dunnett’s test. Each inhibitor reduced the response compared with that of the no inhibitor group (*, P < 0.05). In addition, comparisons between each of the groups treated with all other inhibitors and the group treated with PD98059 demonstrated significantly greater reductions in response compared with that in the group given PD98059 (P < 0.05).

 


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  		Figure 2. Effect of IL-6-type cytokines on the human osteoblast-like MG-63 cell cycle. Cells were cultured in the absence or presence of 50 ng/ml OSM, LIF, IL-11, IL-6, or IL-6 in combination with sIL-6R (1 µg/ml). After 24 h, the distribution of cells in the different phases of the cell cycle was determined by flow cytometry as detailed in Materials and Methods and is expressed as a percentage of the total number of cells. Approximately 10,000 cells were counted for each sample. Similar results were obtained in an additional experiment. Data were analyzed by the {chi}2 test. The {chi}2 statistics vs. control were: for OSM, 2964; for IL-6, 87.67; for IL-6 plus sIL-6R, 2170; for IL-11, 22.11; and for LIF, 31.90. A comparison of OSM and IL-6 plus sIL-6R yielded a {chi}2 value of 84.67. Due to the large number of cells analyzed per condition, all of the {chi}2 statistics (each with 2 degrees of freedom) were significant (P < 0.0001). However, the {chi}2 values involving OSM and IL-6 plus sIL-6R vs. control were approximately 2 orders of magnitude greater than those obtained for the other cytokines vs. control and than that obtained by comparison between OSM and IL-6 plus sIL-6R, indicating major differences in the OSM and IL-6 plus sIL-6R groups compared with the remaining groups.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of IL-6-type cytokines on the proliferation and biosynthetic activity of osteoblastic cells
The results of experiments examining the effects of IL-6-type cytokines on proliferation of the human osteoblast-like MG-63 cell line are summarized in Table 2Go. OSM acted on MG-63 cells to inhibit their proliferation, as indicated by a decrease in [3H]thymidine incorporation (Table 2A) and cell number (Table 2B). This effect was maximal at a cytokine concentration of 10 ng/ml, and it was apparent at 24, 48, and 72 h of culture. Like OSM, IL-6 and IL-11 also inhibited the proliferation of MG-63 cells. LIF, on the other hand, was ineffective (Fig. 1AGo). Similar effects of the cytokines were observed in the presence of 2%, 5%, or 10% serum in the culture medium. Cell viability, as determined by trypan blue exclusion, was not affected in these experiments. In addition, in situ end labeling of fragmented DNA analysis revealed no difference between control and cytokine-treated cells, indicating that the decrease in cell number did not result from an increase in the rate of cell apoptosis. To probe further into the antiproliferative effect of the IL-6-type cytokines on MG-63 cells, we studied their influence on cell cycle progression using flow cytometry. OSM-treated MG-63 cells for 24 h exhibited an increase in the percentage of cells in the G1 phase, and a decrease in the percentage of cells in the S and G2+M phases (Fig. 2Go). IL-6 (or IL-11) by itself did not cause an appreciable change in cell cycle progression. However, when IL-6 was combined with sIL-6R, cell cycle progression was arrested in a manner similar to that seen with OSM. As expected from the experiments on cell proliferation, LIF had no effect on the cell cycle of MG-63 cells.


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  		Table 2. Effect of OSM treatment on MG-63 cell proliferation

 


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  		Figure 1. OSM and other IL-6-type cytokines inhibit proliferation and increase AP activity in human osteoblast-like MG-63 cells. A, Cells were cultured in 2% FBS-MEM in the presence or absence of 10 ng/ml of the indicated cytokines. Thymidine incorporation was performed as detailed in Materials and Methods. Each bar represents the mean of four replicate determinations, expressed as a percentage of the control values. B, Cell monolayers were treated with 10 ng/ml of the indicated cytokines during 4 days. AP activity on cell lysates was determined as detailed in Materials and Methods and expressed as units per mg protein/min (1 U = 10 µg p-nitrophenol). Bars represent the mean ± SD. Data were analyzed by one-way ANOVA. *, P < 0.05 vs. control, as determined by Dunnett’s test.

 
Besides its effect on MG-63 cell proliferation, OSM caused an increase in the level of AP activity. This effect was dose dependent (1–100 ng/ml), with a maximal potency at 10 ng/ml (data not shown). A comparison of the effects of OSM with the effects of other IL-6-type cytokines on AP activity is illustrated in Fig. 1BGo. Compared with OSM, IL-6 had a smaller stimulatory effect on AP activity. Nonetheless, the addition of sIL-6R enhanced the effect of IL-6. On the other hand, neither IL-11 nor LIF had a significant effect on this parameter.

Unlike MG-63 cells, murine osteoblast-like MC3T3-E1 cells as well as primary cultures of murine calvaria cells express the ligand-binding subunits of the receptors for LIF and CNTF (Table 1Go). We, therefore, employed these cells to determine whether LIF and CNTF had effects similar to those of the other members of the cytokine family on osteoblasts. Treatment of MC3T3-E1 cells with either CNTF or LIF caused an increase in the rate of cell proliferation, as evidenced by an increase in the rate of [3H]thymidine incorporation and cell number. This effect was seen as early as 24 h, reached a maximum of 40% over control values by the third day of a 4-day culture period, and was obtained when the cells were cultured in medium contained 2%, 5%, or 10% serum. The proliferation of primary cultures of calvaria cells, on the other hand, was not affected by treatment with either cytokine (data not shown).

Similar to the effect of other cytokines on AP activity in MG-63 cells, CNTF and LIF increased the levels of AP activity in MC3T3-E1 and calvaria cells (Fig. 3Go). The effect of either cytokine was dose dependent over a concentration range of 0.1–50 ng/ml (data not shown). The magnitude of the cytokine-induced AP increases at 10 ng/ml in MC3T3-E1 cells was similar to that induced by 10-8 M 1,25(OH)2D3 (Fig. 3AGo). That the increased AP activity was the result of increased expression of the AP gene was strongly suggested by Northern blot analysis, which showed a dose-dependent increase in the abundance of the AP mRNA in response to both LIF and CNTF in MC3T3-E1 cells (Table 3Go). The increase in AP mRNA in cells maintained in the presence of CNTF and LIF was accompanied by a similar increase in the abundance of another osteoblast phenotypic marker mRNA, namely osteocalcin.



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  		Figure 3. CNTF and LIF increase AP activity in murine osteoblast-like MC3T3 cells and primary cultures of murine calvaria cells. Cells were plated at 5 x 103/cm2 and cultured in 2% FBS-MEM in the presence or absence of 10 ng/ml CNTF, 10 ng/ml LIF, or 10-8 M 1,25-(OH)2D3 (D3; four replicates per condition). AP activity in cell lysates was performed using p-nitrophenyl phosphate as a substrate and corrected for protein content. Bars represent the mean ± SD. Data were analyzed by ANOVA. *, P < 005 vs. control (by Dunnett’s test).

 

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  		Table 3. Effects of LIF or CNTF on the expression of mRNAs for osteocalcin and AP in MC3T3-E1 cells

 
Primary cultures of calvaria cells also exhibited increased AP activity when treated with CNTF or LIF (Fig. 3BGo). Addition of soluble CNTF receptor did not lead to any further increase in the effect of CNTF alone. This finding is in agreement with our earlier studies indicating that the amount of CNTF receptor {alpha} expressed in calvaria cells is sufficient for a maximal response to this cytokine (12). IL-11 also increased AP activity in calvaria cells, whereas IL-6 was ineffective. These results are in agreement with the earlier evidence that neonatal murine calvaria cells express receptors for IL-11, but not for IL-6 (Table 1Go).

In agreement with earlier evidence that OSM, LIF, and CNTF increase the production of IL-6 in cell types other than osteoblasts, OSM increased the production of IL-6 in MG-63 cells, and LIF and CNTF increased IL-6 production in MC3T3-E1 cells (data not shown).

IL-6-type cytokines activate both the JAK/STAT and the MAPK pathways in osteoblastic cells
Having demonstrated the effects of IL-6-type cytokines on the biosynthetic activity and the rate of cell proliferation of osteoblastic cells, and the strict correspondence of these effects with the presence of the {alpha}-subunit of the cytokine receptor, we next sought to determine the signaling pathways responsible for the mediation of these effects. Short term stimulation (15 min) of MG-63 cells with IL-6-type cytokines caused the formation of three protein complexes that retarded the mobility of a radiolabeled oligonucleotide probe containing a known STAT-binding element (SBE) in EMSA (Fig. 4AGo). The abundance of the protein-DNA complexes formed in response to different cytokines correlated closely with the concentration of the respective cytokine receptor, determined in our earlier studies (12). Thus, OSM induced the strongest activation of STATs, followed by IL-6 in combination with sIL-6R, IL-6 alone, and IL-11. LIF, on the other hand, failed to induce STAT activation in MG-63 cells. The presence of STAT1{alpha} and STAT3 in the complexes was established by supershift analysis (Fig. 4BGo). Thus, an antibody against STAT1{alpha} inhibited the formation of both the fastest and the intermediate migrating complexes, whereas an anti-STAT3 antibody supershifted both the slowest and the intermediate complexes. STAT complexes identical to those identified in MG-63 cells in response to OSM were activated in MC3T3-E1 cells in response to LIF, CNTF, or CNTF in combination with its soluble receptor.



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  		Figure 4. IL-6-type cytokines induce the formation of STAT complexes in osteoblastic cells. A, Semiconfluent cultures of MG-63 cells were stimulated with 50 ng/ml OSM, LIF, IL-11, and IL-6 alone or in combination with 1 µg/ml sIL-6R for 15 min. Nuclear extracts were obtained, and EMSA was performed as described in Materials and Methods. The probe was incubated with nuclear proteins from unstimulated cells (Basal) or cells stimulated with the indicated cytokines in the absence (lines 1, 3, 5, 7, 9, and 11) or presence (lines 2, 4, 6, 8, 10, and 12) of a 100-fold molar excess of unlabeled oligonucleotide. B, Supershifts were performed by incubating the nuclear proteins with 1 µg nonimmune IgG (ni), anti-STAT1{alpha}, or anti-STAT3 antibody for 20 min at room temperature before addition of the probe. Arrows indicate the positions of STAT3 homodimers, STAT3/STAT1{alpha} heterodimers, and STAT1{alpha} homodimers.

 
In addition to activating STATs, stimulation of osteoblastic cells with IL-6-type cytokines activated the MAPK pathway. Indeed, 15-min exposure of MG-63 cells to OSM and IL-6 plus sIL-6R (but not to IL-6 alone, IL-11, or LIF) caused an increase in MAPK activity, as evidenced by the ability of cell lysates to phosphorylate MBP, a known substrate of MAPK, in an in-gel kinase assay (Fig. 5AGo). The increase in MAPK activity was associated with an increase in the amount of tyrosine-phosphorylated MAPK, as determined by Western blot analysis with an antiphospho-MAPK antibody. However, immunoblot analysis with an anti-MAPK antibody indicated that the concentration of unphosphorylated MAPK did not change upon stimulation of MG-63 cells with OSM or IL-6 plus sIL-6R (Fig. 5BGo). The time course of the effects of OSM on MAPK activity and phosphorylation is shown in Fig. 6Go, A and B, respectively. The effect of the cytokine reached a peak within 5–15 min and decreased significantly by 30 min. Although the Western blots for phosphorylated MAPK seem to be more sensitive in detecting activation of MAPK than the in-gel kinase assay, the two techniques produced similar results.



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  		Figure 5. IL-6-type cytokines increase the activity of MAPK in osteoblastic cells. MG-63 cells were stimulated with the cytokines alone (50 ng/ml) or with IL-6 in combination with its soluble receptor (1 µg/ml) for 15 min and lysed as detailed in Materials and Methods. A, MAPK activity in cell lysates was assayed by an in-gel kinase assay. Proteins (50 µg/line) were electrophoresed on SDS-polyacrylamide gels containing MBP, and the kinase reaction was performed in the gel. Lysates from insulin-like growth factor I-stimulated NFB4 cells (30 µg) were included as a positive control for Erk activation (+C). B, The level of tyrosine-phosphorylated MAPK (Erk1 and -2) in untreated or cytokine-stimulated MG-63 cells was determined by Western blot analysis using an antiphospho-MAPK antibody (upper panel) and an anti-MAPK antibody (lower panel). Proteins from PMA-stimulated MG-63 cells were included in the same gel as a positive control for MAPK phosphorylation. Purified phospho-MAPK (phos MAPK) and unphosphorylated MAPK (unphos MAPK) control proteins were also run.

 


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  		Figure 6. Time response of the activation of MAPK by OSM. MG-63 cells were stimulated with OSM (50 ng/ml) for the indicated times and lysed as detailed in Fig. 5Go. A, The kinase activity of the cell lysates was assayed by an in-gel kinase assay. B, The level of tyrosine-phosphorylated MAPK (Erk1 and -2) in untreated or OSM-stimulated MG-63 cells was determined by Western blot analysis using an anti-phospho-MAPK antibody (upper panel) and an anti-MAPK antibody (lower panel), as described in Fig. 5Go. The ratios of phospho-MAPK/MAPK were obtained by quantifying the intensity of the bands in the autoradiographies using a laser densitometer.

 
Tyrosine and serine/threonine kinase inhibitors block the effects of OSM on osteoblastic cells
The relative contributions of the JAK/STAT and MAPK pathways to the prodifferentiating and antiproliferative effects of OSM on MG-63 cells were evaluated using the tyrosine kinase inhibitors staurosporine and herbimycin A, the threonine/serine kinase inhibitor H7, and PD98059, an inhibitor of the tyrosine kinase MEK. As it has been recently documented (29, 30, 31), PD98059 is specific inhibitor of MEK, which is, in turn, responsible for MAPK activation. Staurosporine as well as H7 completely blocked the effect of OSM on cell cycle progression (Fig. 7AGo). In addition, they abolished the stimulatory effect of OSM on the AP activity of MG-63 cells (Fig. 7BGo). Similar results were obtained using herbimycin A, another tyrosine kinase inhibitor (not shown). None of the kinase inhibitors affected cell viability in these experiments, as evidenced by trypan blue exclusion. In addition, new protein synthesis inhibition by cycloheximide blocked the effects of OSM in these cells without affecting cell viability.

In contrast to the ability of inhibitors of tyrosine and threonine/serine kinases to abrogate the effects of OSM in MG-63 cells, the MAPK inhibitor PD98059 did not influence the effects of OSM on either cell proliferation or AP activity (Fig. 7Go). PD98059 in these experiments was used at 5–10 µM, which correspond to the IC50 for the inhibition of MEK activation (30, 31). However, when PD98059 was used in these experiments at 50 µM, it slightly decreased the effects of OSM (Fig. 7Go). The effects of the inhibitors on the activation of STATs by OSM were monitored by EMSA using as a probe the same SBE employed in Fig. 4Go (Fig. 8AGo). Pretreatment of the cells with the tyrosine kinase inhibitor staurosporine abolished completely the ability of OSM to induce STAT binding to the SBE probe. On the other hand, nuclear extracts from MG-63 cells stimulated with OSM and pretreated with cycloheximide, H7, or PD98059 showed no significant differences in STAT binding to the SBE probe compared with cells stimulated with OSM alone. This is consistent with the idea that cytokines increase the DNA-binding activity of STATs, inducing the phosphorylation of preexisting STAT molecules (5), and that serine phosphorylation is required for the maximal transcriptional activity of STATs, but is not essential for DNA binding in vitro (6). Nonetheless, PD98059 completely suppressed the OSM-induced increases in MAPK phosphorylation, as determined by immunoblotting with a phospho-MAPK antibody (Fig. 8BGo).



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  		Figure 8. Specificity of the effect of PD98059 on MAPK activation. A, EMSA were performed as described in Fig. 4Go, except that MG-63 cells were treated with 1 µM cycloheximide, 0.1 µM staurosporine, 50 µM H7, or 50 µM PD98059 for 30 min before the addition of OSM. B, Cells were treated with vehicle (None) or with 50 µM PD98059 for 30 min before the addition of 50 ng/ml OSM for 15 min. Monolayers were washed and lysed, and proteins (100 µg/lane) were separated by SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. The level of phosphorylated MAPK (Erk1 and -2) was determined by Western blot analysis using an antiphospho-MAPK antibody as described in Figs. 5BGo and 6BGo. These results were reproduced in two additional experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of the experiments presented here demonstrate that IL-6-type cytokines act on osteoblastic cells to stimulate AP and osteocalcin, two routinely used and widely accepted phenotypic markers of osteoblast differentiation (32, 33, 34). Stimulation of markers of osteoblast differentiation in our studies was a consistent finding in both murine and human osteoblastic cell lines as well as in primary cultures of osteoblastic cells, even though the effects of the cytokines on cell proliferation varied. In full agreement with the findings of this report, other investigators found that LIF potentiates the expression of AP and collagen in osteoblastic cells (35, 36). In addition, studies of ours reported elsewhere demonstrate that IL-6 plus sIL-6R or LIF stimulate the differentiation of uncommitted mesenchymal progenitors of the bone marrow toward the osteoblast phenotype (37, 38), and that IL-6 plus sIL-6R or IL-11 act on embryonic fibroblasts (12–14 days gestation) to promote commitment toward the osteoblast phenotype, without affecting the differentiation toward adipocytes or muscle cells (39).

As shown previously in other cell types, IL-6-type cytokines acted on osteoblastic cells to activate both the JAK/STAT and MAPK pathways. However, the results of our experiments with specific kinase inhibitors suggest that JAK/STAT activation is critical for the effects of the cytokines on osteoblast proliferation and differentiation, whereas MAPK activation may play a minimal role in these particular effects. Thus, PD98059 specifically blocked MAPK phosphorylation, whereas it had a minimal influence on the effects of OSM on AP or cell proliferation and did not alter STAT-binding activity. We are quick to point out, however, that from these results, we cannot exclude the possibility that the MAPK pathway can be indirectly involved in the prodifferentiating effects of IL-6-type cytokines by modulating the transcriptional activity (as opposed to the DNA binding) of STATs. Such a scenario could account for the small inhibitory effect of high concentrations of PD98059 on OSM responses. Be this as it may, future studies using dominant negative constructs of JAK/STAT and MAPK would be required to provide definitive evidence for or against the importance of the MAPK pathway in these responses.

Although all the cytokines had the same qualitative effects, the ability of a particular cell type to respond to a given cytokine was strictly dependent on the presence of the ligand-binding subunit of the receptor, and the magnitude of the effect correlated closely with the concentration of this subunit. This evidence demonstrates that the ligand-binding subunits of the IL-6 type cytokines are the determining factors for their biological relevance to bone metabolism (11). It has been proposed that sequences present in the ß-subunits of the cytokine receptors determine the activation of specific substrates (40). Therefore, distinct biological effects of cytokines of this group may result from the presence of a different ß-subunit in the receptor complexes. Our observations that each member of the IL-6-type cytokine family had similar effects regardless of whether the cytokine induces homodimerization of gp130 (IL-6 and IL-11) or heterodimerization of gp130 with other ß-subunits (LIF, CNTF, and OSM) do not support this contention. Nonetheless, as we have only examined the effects of IL-6-type cytokines on cell proliferation, AP activity, and osteocalcin production, we cannot exclude the possibility that different members of this group of cytokines may elicit different biological responses from other aspects of osteoblast biology. Similarly, we cannot exclude the possibility that activation of the MAPK pathway in osteoblastic cells is responsible for cytokine effects other than those we monitored here.

Whereas IL-6-type cytokines inhibited the rate of proliferation of human MG-63 cells, they had a stimulatory effect on the proliferation of murine MC3T3-E1 cells and no effect at all on the proliferation of murine calvaria cells. These differences were consistently observed in subconfluent cultures and were confirmed in several repeat experiments regardless of whether they were conducted in 2%, 5%, or 10% serum. Similar to our observations, it has been previously shown that LIF can either inhibit or stimulate proliferation in different osteoblastic cell types (41, 42, 43, 44). At this stage, it is unknown whether these differences are due to differences between homogeneous (MC3T3-E1) and heterogeneous (primary calvaria) cell preparations, the stage of differentiation represented by a given cell preparation, or even inherent differences in the proliferative potential of transformed cell lines. However, it is important to note that although proliferation and differentiation have in general a reciprocal relationship both in vitro and in vivo, in vitro the expression of differentiated function and proliferation occur at the same time, whereas in vivo there is a clear temporal and spatial separation between them (34, 45, 46).

The significance of the in vitro evidence for prodifferentiating effects of IL-6-type cytokines on cells of the osteoblastic lineage to the in vivo situation is a matter of conjecture. Nonetheless, there is considerable evidence to suggest that the prodifferentiating effects of IL-6-type cytokines on cells of the osteoblastic lineage may be intricately linked to the osteoclastogenic properties of these cytokines. Thus, it is well documented that the osteoclastogenic effects of IL-6-type cytokines cannot be manifested in vitro unless osteoblastic cells are present (47). Taken together with the results of the present report and other supporting evidence discussed above, this evidence points to the possibility that IL-6-type cytokines promote osteoclast development at least in part by promoting the differentiation of stromal/osteoblastic cells that provide support for osteoclastogenesis. Strong support for this concept has been recently provided by evidence that loss of sex steroids (which is thought to cause loss of bone because of increased production of IL-6) does not cause the expected increase in cancellous osteoclasts and trabecular spacing, nor does it cause the expected decrease in bone mineral density in an animal model of defective osteoblast development (48). Several lines of evidence have suggested that besides their osteoclastogenic and bone-resorbing effects, IL-6-type cytokines have bone-forming properties as well. Indeed, it has been shown that mice overexpressing LIF exhibit sclerotic bone marrow with excessive woven bone as well as ectopic bone formation (49). Similarly, mice overexpressing OSM exhibit increased bone formation and enlarged hind limbs (50). Conversely, targeted disruption of the LIF receptor ß gene results in decreased bone volume in the primary spongiosa of developing bone of fetal mice (51). The in vitro evidence for prodifferentiating effects of IL-6-type cytokines on cells of the osteoblastic lineage, discussed here, provides a mechanistic explanation for these observations, and it is in line with the contention that IL-6-type cytokines are capable of regulating both osteoclastogenesis and osteoblastogenesis (11). In fact, stimulation of both osteoclastogenesis and osteoblastogenesis by IL-6-type cytokines can explain the fact that the majority of osteoclasts and osteoblasts are needed in the progression of the basic multicellular unit during bone remodeling, and they are needed simultaneously, osteoclasts at the apex and osteoblasts at the deepest portion of the cutting cone.

Finally, our earlier observations that different osteoblastic cells express different receptor repertoires for these cytokines have led us to speculate that the expression of a particular cytokine receptor could be a function of the state of osteoblast differentiation (12). If that were the case, the present finding that all members of the group had similar prodifferentiating effects raises questions vis à vis the teleological significance of a differentiation-dependent expression or repression of a given receptor. Nonetheless, it is theoretically possible that as the basic multicellular unit moves across the bone surface (52), an osteoblast precursor cell will be exposed to different microenvironments (i.e. cytokine soups). A changing cytokine receptor repertoire, therefore, will allow the differentiating osteoblast to respond in a manner that anticipates the need for fully differentiated osteoblasts to arrive at a specific time and place on the bone surface.


   Acknowledgments
 
The authors thank Li Han for excellent technical assistance; J. Woodliff and Dr. J. Epstein for the flow cytometric analysis; Dos Sarbassov for the gift of insulin-like growth factor I-stimulated lysates from NFB4 muscle cells; the Arkansas Cancer Research Center Office of Grants and Scientific Publications for editorial support; and Drs. N. Stahl, R. L. Jilka, and A. M. Parfitt for very helpful discussions.


   Footnotes
 
1 This work was supported by the NIH (Grant PO1AG/AMS13918; to S.C.M. and Grant R29AR43453 to T.B.), the V.A., and a University of Arkansas for Medical Sciences faculty development grant and an American Cancer Society Institutional Grant (187-B; to T.B.). Back

Received February 3, 1997.


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 Introduction
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 Results
 Discussion
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