Endocrinology Vol. 138, No. 9 3666-3676
Copyright © 1997 by The Endocrine Society
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
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Abstract
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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 (
) 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.
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Introduction
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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 (
-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, Pagets 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
-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
- and ß-subunits of the
receptors for IL-6, IL-11, LIF, CNTF, and OSM. However, different
repertoires of
- 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
(
-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.
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Materials and Methods
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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
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 (12 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 1
. 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
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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 0.750.9 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 14 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 8590% confluence (3648 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
8590% 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 8590% 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 8590% 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, 47 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 [
-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
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
[
-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 manufacturers
recommendations (New England Biolabs).
Statistical analysis
Data were analyzed by one-way ANOVA, and Dunnetts 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. 7
), 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 Dunnetts 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 Dunnetts 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. 2
) were analyzed using the
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. 2 . 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 Dunnetts 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 2 test.
The 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 2 value of 84.67. Due to the large number of
cells analyzed per condition, all of the 2 statistics
(each with 2 degrees of freedom) were significant
(P < 0.0001). However, the 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.
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Results
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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 2
. 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. 1A
).
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. 2
). 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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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 Dunnetts test.
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Besides its effect on MG-63 cell proliferation, OSM caused an increase
in the level of AP activity. This effect was dose dependent (1100
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. 1B
. 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 1
). 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. 3
). The effect of either cytokine
was dose dependent over a concentration range of 0.150 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. 3A
). 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 3
). 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 Dunnetts test).
|
|
Primary cultures of calvaria cells also exhibited increased AP activity
when treated with CNTF or LIF (Fig. 3B
). 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
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 1
).
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
-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. 4A
). 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
and STAT3 in the complexes was
established by supershift analysis (Fig. 4B
). Thus, an antibody against
STAT1
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 , or anti-STAT3 antibody for 20 min at room temperature
before addition of the probe. Arrows indicate the
positions of STAT3 homodimers, STAT3/STAT1 heterodimers, and
STAT1 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. 5A
). 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. 5B
). The time course of the effects of OSM on MAPK
activity and phosphorylation is shown in Fig. 6
, A and
B, respectively. The effect of the cytokine reached a peak within 515
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.
|
|
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. 7A
). In addition, they abolished the
stimulatory effect of OSM on the AP activity of MG-63 cells (Fig. 7B
).
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. 7
). PD98059 in these
experiments was used at 510 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. 7
).
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. 4
(Fig. 8A
). 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. 8B
).
 |
Discussion
|
|---|
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
(1214 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.). 
Received February 3, 1997.
 |
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