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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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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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) 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 |
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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, 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 -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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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 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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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 [
-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 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. 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 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. 2) were analyzed using the
2 test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. 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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. 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 0.1–50 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. 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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). 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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). 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 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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). 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 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. 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).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Received February 3, 1997.
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