From the Division of Endocrinology and Metabolism, the Center for Osteoporosis and Metabolic Bone Diseases, University of Arkansas for Medical Sciences, and the McClellan Veterans Affairs Medical Center, Geriatric Research, Education, and Clinical Center, Little Rock, Arkansas 72205
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ABSTRACT |
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The cyclin-dependent kinase inhibitor p21WAF1,CIP1,SDI1 plays a critical role in cell differentiation, and it has been shown to confer resistance to apoptosis. Based on this, and on evidence that activation of the gp130/signal transducer and activator of transcription (STAT) signal transduction pathway by interleukin (IL)-6 type cytokines promotes differentiation and prevents apoptosis in osteoblastic cells, we have investigated the possibility that p21 is a downstream effector of this signaling pathway in osteoblasts. We report that either oncostatin M (OSM) or IL-6 plus soluble IL-6 receptor increased the levels of p21 mRNA and protein in the osteoblast-like human osteosarcoma cell line MG63 and stimulated the activity of a 2.4-kilobase pair segment of the human p21 gene promoter. Further, nuclear extracts from cytokine-stimulated MG63 cells formed protein-DNA complexes with a 19-base pair nucleotide fragment of the p21 promoter containing a single STAT response element. The identity of the binding proteins as Stat3 and Stat1 was demonstrated with specific antibodies. In addition, and in support of a mediating role of STATs in the activation of the p21 promoter, overexpression of Stat3 potentiated the cytokine effect on the p21 promoter; whereas a dominant negative Stat3, or a mutation of the STAT response element on the promoter, significantly reduced the cytokine effect. Finally, antisense oligonucleotides complementary to p21 mRNA inhibited OSM-induced stimulation of alkaline phosphatase expression and antagonized the protective effect of OSM on anti-Fas-induced apoptosis. These results demonstrate that p21 is a downstream effector of gp130/Stat3 activation and a critical mediator of the pro-differentiating and anti-apoptotic effects of IL-6 type cytokines on human osteoblastic cells.
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INTRODUCTION |
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Fine orchestration of the activity of cyclins, Cdk,1 and inhibitors of such kinases governs the progression of cells from one phase of their division cycle to the next. Increased expression of Cdk inhibitors is now recognized as a general mechanism for the arrest of cell division (1, 2). One of these Cdk inhibitors, designated as p21WAF1,CIP1,SDI1 because of its original identification by three independent groups as wild type p53 activated fragment 1 (WAF1) (3), Cdk-interacting protein 1 (CIP1) (4), or senescence cell-derived inhibitor (SDI1) (5), binds and inactivates several cyclin/Cdk complexes including the ones involved in the transition between G1 and S phases, and inhibits the replication factor PCNA (proliferating cell nuclear antigen) (6). To date, p21 has been implicated in three different processes: DNA damage repair, differentiation, and apoptosis. In the former, p21 gene expression is stimulated by the tumor suppressor gene p53, and the protein arrests cells in the G1 phase in order to allow the DNA damage repair process to be performed before cells reenter the S phase (7, 8). In the latter two processes, p21 expression increases independently of p53. In this case, the resulting halt of cell division allows cells to progress along their differentiation pathway (9-14). Interestingly, in differentiating muscle cells, neuroblastoma, and melanoma cells, p21 also protects against programmed cell death (15-17).
It is now established that cytokines such as IL-6 can influence bone resorption and formation by regulating the production of osteoclasts and osteoblasts (reviewed in Ref. 18). Indeed, IL-6, IL-11, LIF, and OSM are produced by cells of the stromal/osteoblastic lineage in response to local factors and systemic hormones, and they stimulate osteoclast differentiation from hematopoietic precursors. In addition, they modulate the rate of proliferation and promote the differentiation of osteoblast progenitors and committed osteoblastic cells from rodent and human origin (18-21). Moreover, IL-6 type cytokines suppress osteoblastic cell apoptosis induced by serum withdrawal, glucocorticoids, tumor necrosis factor, or the Fas pathway (22, 23).
The receptors for all these cytokines share a common signal-transducing component known as gp130 (24). Ligand-induced dimerization of gp130 initiates intracellular signaling by activating members of the Janus kinase family of tyrosine kinases (25). This step induces the recruitment, tyrosine phosphorylation, and nuclear translocation of STATs, which activate transcription of cytokine-responsive genes (26, 27).
Because of evidence that p21WAF1,CIP1,SDI1 is induced during
cell differentiation and confers resistance to apoptosis in some cell
types, we have investigated whether this gene is regulated by IL-6 type cytokines and whether it plays a role in their actions on osteoblasts. For these studies, we utilized an established human osteosarcoma cell
line, MG63, which exhibits many features of normal osteoblastic cells
and has been used extensively as a model of this cell type (28). MG63
cells express high levels of the OSM receptor and low levels of the
ligand binding (
) subunit of the IL-6 receptor (29); hence, the
gp130 signal pathway can be activated by OSM or by the combination of
IL-6 with sIL-6R. We have previously reported that such activation
leads to cell cycle arrest and a simultaneous increase in the
expression of the phenotypic marker AP (19), and prevents Fas-induced
apoptosis (22). Here, we present evidence that activation of the
gp130/STAT signal transduction pathway by IL-6 type cytokines leads to
the transcriptional activation of the p21WAF1,CIP1,SDI1 gene,
and that p21 is a critical mediator of the differentiation-promoting and anti-apoptotic actions of these cytokines on osteoblastic cells.
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EXPERIMENTAL PROCEDURES |
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Materials--
Human recombinant IL-6 was obtained from Upstate
Biotechnology Inc. (Lake Placid, NY). sIL-6R and human recombinant OSM
were from R&D Systems (Minneapolis, MN). FBS, L-(+)-lactic
acid, diaphorase, AP buffer, p-nitrophenyl phosphate (Sigma
104), and monoclonal anti--actin antibody (clone AC-74, IgG2a) were
purchased from Sigma. MEM and Lipofectin (1:1 (wt/wt) mixture of
N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride and dioleoyl phosphatidylethanolamine) were from Life Technologies, Inc. MTS was from Promega (Madison, WI). Mouse monoclonal anti-Stat1
, rabbit polyclonal anti-Stat3, goat polyclonal anti-p21, mouse monoclonal anti-p53, anti-mouse IgG-HRP, and anti-goat IgG-HRP antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz,
CA). ECL reagents were purchased from NEN Life Science Products. Phosphorothioate oligodeoxynucleotides were from Cruachem Inc. (Dulles,
VA). Human p21 cDNA was kindly provided by Dr. James R. Smith
(Baylor College of Medicine, Houston, TX) (5). Human
-actin cDNA
was obtained from CLONTECH.The human p21 promoter in pGL2 plasmid (Promega) was provided by Dr. Leonard Freedman (Memorial Sloan-Kettering Cancer Center, New York, NY) (11). Finally,
the expression vectors containing the cDNA for human Stat3 and
dominant negative Stat3 (Y705) were kindly provided by Dr. Eric
Caldenhoven (University Hospital Utrecht, Utrecht, The Netherlands)
(30) and Dr. Michael Saunders (Glaxo-Wellcome, Les Ulis Cedex, France)
(31), respectively.
Cell Culture Conditions-- Human osteosarcoma MG63 cells (32) were cultured in phenol red-free MEM supplemented with 10% FBS, 100 units/ml penicillin, and 100 µg/ml streptomycin. Cultures were kept in a humidified atmosphere of 5% CO2 in air at 37 °C.
Western Blot Analysis--
Cells were lysed in 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 unit/ml aprotinin, 1 mM
phenylmethylsulfonyl fluoride, and 1% Triton X-100. Insoluble material
was pelleted in a microcentrifuge at 10,000 × g for 10 min. Protein lysates were dissolved in buffer for protein
electrophoresis, separated on SDS-polyacrylamide gels, and
electrotransferred to polyvinylidene difluoride. 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 an antibody to either p21, p53, or -actin, followed by
incubation for 1 h with the corresponding secondary antibody conjugated with HRP. Blots were developed by ECL, according to the
manufacturer's recommendations. Quantification of the intensity of the
bands in the autoradiograms was performed by laser densitometry.
RNA Extraction and Northern Blot Analysis-- Total cellular RNA was isolated from semiconfluent control or cytokine-treated cultures, and polyadenylated RNA was selected as described previously (29). Messenger RNA 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 cDNAs for p21 or for actin, and analyzed using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Electrophoretic Mobility Shift Assays--
Electrophoretic
mobility shift assays were performed essentially as described elsewhere
(19). Eighty to 90% confluent cultures of MG63 cells were maintained
in serum-free medium for 2 h and stimulated with 50 ng/ml OSM for
15 min at 37 °C. Cells were rinsed once with PBS, and nuclear
extracts were prepared. Cells (4-7 × 107) were
washed with hypotonic buffer (10 mM Hepes, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol) and were 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 × 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 dithiothreitol, and 25%
glycerol) and were incubated at 4 °C for 15 min. Lysed nuclei were
dispersed and centrifuged at 10,000 × g at 4 °C for
10 min. Supernatants were collected, snap frozen, and stored at
70 °C. A synthetic double-stranded oligonucleotide corresponding
to the most proximal Stat binding element (p21-SIE1: -640 nucleotides
from the TATA promoter site) present in the p21 promoter
(5'-GATCTCCTTCCCGGAAGCA-3') (3, 33) was end-labeled using
[
-32P]ATP and T4 polynucleotide kinase. The probe (1 ng) was incubated for 20 min at room temperature with 10 µg of
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 100 × molar excess unlabeled SIE1
wild type oligonucleotide or unlabeled mutated SIE1 (SIE1M: 5'-GATCTCCAAGCTTGAAGCA-3'). Supershift experiments were performed by incubating the nuclear proteins with either non-immune IgG, anti-Stat1
antibody, or anti-Stat3 antibody, for 10 min at room
temperature prior to the addition of the labeled probe.
Transient Transfections, Assay of Reporter Gene Activity, and
Oligonucleotide-directed Mutagenesis--
Transient transfections of
MG63 cells were carried out in 12-well culture plates using SuperFect
transfection reagent (Qiagen) and the dual-luciferase reporter assay
system (Promega). Cells were transfected with 2.5-3 µg of a p21-luc
reporter construct containing a 2.4-kb segment of the human p21 gene
promoter (or an identical segment in which the putative STAT binding
segment was mutated as described below) inserted into the firefly
luciferase reporter gene, pGL2-basic (3, 11), together with 3 ng of the
co-reporter vector containing the Renilla luciferase gene and the CMV immediate early enhancer/promoter region (pRL-CMV). Reporter plasmids were cotransfected with 0.5 µg of the expression plasmid for Stat3 or the empty vector (30). Forty-eight hours after
transfection, cells were treated with OSM (20 ng/ml) for the indicated
times, and dual luciferase activity was analyzed in cell lysates
according to the manufacturer's instructions. Light intensity was
measured with a Turner luminometer, and firefly luciferase activity was
normalized versus Renilla luciferase activity. For oligonucleotide-directed mutagenesis, a 30-base oligonucleotide was
synthesized as the reverse complement of the p21 sense strand containing a mutation in the SIE1 sequence (660 nucleotides from the
TATA promoter site: 5'-GTCACATGCTTCAAGCTTGGAGGGAATTGG-3'). This was used to anneal and extend the wild type single-stranded template generated from p21-luc, a plasmid containing a 2.4-kb HindIII segment of the human p21 gene promoter inserted into
the firefly luciferase reporter gene, pGL2-basic (11). The mutation incorporated a HindIII restriction site that was used for
the selection of mutated plasmids, and it was confirmed by sequencing (34).
Antisense Oligonucleotides--
Phosphorothioate
oligodeoxynucleotides (15.7 µM) and Lipofectin (3 × 104 mg/ml) were incubated at 37 °C for 15 min. The
mixture was diluted with serum-containing medium to a final
concentration of 1 µM oligonucleotides, and added to the
cells. Antisense oligonucleotides were based on the
p21WAF1,CIP1,SDI1 coding sequence. AS-IC is complementary to
the region around the initiation codon (5'-TCC CCA GCC GGT TCT GAC
AT-3'), AS-MID is complementary to the middle of the coding region
(5'-CCT CCA GTG GTG TCT CGG TG-3'), AS-3' is complementary to the 3'
end (5'-TGT CAT GCT GGT CTG CCG CC-3'), and the control sense
oligonucleotide S-IC is complementary to the AS-IC (5'-ATG TCA GAA CCG
GCT GGG GA-3').
AP Activity--
AP activity was determined as previously
reported (19). MG63 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 cells
were cultured with or without 20 ng/ml OSM in the absence or presence
of 1 µM phosphorothioate oligonucleotides for 3 days (8 replicas per condition). 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 LDH, using a microtiter plate reader. Monolayers were
washed once with distilled water and three times with 0.2 M
Tris, pH 8.2. 75 µl of 0.2 M Tris, pH 8.2, was then added
to each well. Subsequently, 75 µl of 0.2 M Tris, pH 8.2, containing 1.3 × 103 M NAD, 5.4 × 10
2 M L-(+)-lactic acid, 6 mg/ml
MTS, and 2.572 units/ml diaphorase, was added to each well, and
readings at 490 nm and at 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 of AP buffer and 75 µl of 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/min)
to MTS/LDH rate (OD/min).
Apoptosis--
Apoptosis of human osteoblast-like MG63 cells was
induced as described previously (22). Cells were cultured for 24 h
in MEM containing 10% FBS in the absence or presence of 0.1 nM -IFN, alone or in combination with 20 ng/ml OSM. The
medium was then replaced with 10% FBS-MEM containing 10 µg/ml
anti-Fas antibody with or without OSM. Cells were cultured for an
additional 16 h, and apoptotic cells were quantified by trypan
blue staining. Nonadherent cells were combined with adherent cells
released from the culture dish using trypsin-EDTA, and resuspended in
PBS. Subsequently, 0.4% trypan blue was added, and the percentage of
cells exhibiting both nuclear and cytoplasmic staining was determined
using a hemocytometer. At least 200 cells per condition were counted.
We have previously demonstrated that the percentage of cells exhibiting
trypan blue staining correlates with the percentage of TUNEL-labeled
cells, indicating that apoptotic osteoblasts could be reliably
quantified by either method (22).
Statistical Analysis-- For the statistical analysis of the data of Fig. 7, standardized means and standard errors were calculated using the variance for the ratio of two random variables (35). Approximate ninety-five percent confidence intervals for the standardized means of the OSM-treated groups were calculated as the standardized mean ± 2 standard errors. Confidence bounds with a lower limit greater than 1 were considered to denote a significant (p < 0.05) increase over the corresponding control group. For the analysis of the data regarding the effects of OSM on AP in the presence of oligonucleotides (Fig. 8B), 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 oligonucleotide group was compared with the group without oligonucleotide using the Dunnet's test and an experiment-wise significance level of 0.05. In addition, an analysis using Dunnet's test to compare all antisense oligonucleotide groups to the group treated with sense oligonucleotide was also conducted. Data of Fig. 8C were analyzed by one-way ANOVA, and Dunnet's test was utilized to estimate the level of significance of differences between means.
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RESULTS |
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IL-6 Type Cytokines Increase the Protein Levels of p21 in a p53-independent Fashion-- MG63 cells cultured in the presence of OSM exhibited an increase in the levels of p21 protein, as detected by Western blot analysis (Fig. 1A). This effect was evident as early as 2 h of treatment and remained present for at least 24 h. By 48 h, p21 levels had returned to base-line levels. Similar increases in p21 protein expression were found when IL-6 in combination with sIL-6R was used in these experiments instead of OSM (Fig. 1B). However, there was no change in the levels of p53 protein in cells treated with OSM (Fig. 1A). Consistent with the results of the Western blot analysis, p21 protein was detected by immunohistochemistry in MG63 cells maintained under basal conditions and OSM increased the staining (Fig. 2, upper panels). Nuclear staining was abolished when preparations were incubated with the anti-p21 antibody together with the peptide to which the antibody was raised (lower panels).
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gp130 Activation Increases Steady State p21 mRNA Levels-- We next examined p21 mRNA expression in control and OSM-treated MG63 cells. As shown in Fig. 4A, OSM increased the abundance of p21 mRNA as early as 1 h after treatment. The effect was maximal at 6 h, decreased at 24 h, and returned to control levels by 48 h. To investigate whether de novo protein synthesis was required, we examined the regulation of p21 mRNA in the presence of cycloheximide. In this experiment, cells were preincubated with cycloheximide for 30 min, and then treated with OSM for 6 h. Cycloheximide did not block the effect of OSM. In fact, both basal and OSM-induced p21 levels were significantly increased in the presence of cycloheximide (Fig. 4B), suggesting that p21 mRNA levels may be posttranscriptionally regulated by labile proteins.
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gp130 Activation Increases the Transcriptional Activity of the p21 Promoter, and Stat3 Potentiates This Effect-- To determine whether gp130 activation affected the transcriptional activity of the p21 promoter, MG63 cells were transiently transfected with a 2.4-kb fragment of the p21 promoter inserted upstream from the luciferase reporter gene. Cells were then treated with OSM and luciferase activity was determined at different time points following the addition of the cytokine (Fig. 6A). OSM increased luciferase activity, with a maximal effect after 6 h of treatment. The effect of OSM was greater in cells transfected with the promoter/luciferase construct together with a Stat3 expression plasmid. The luciferase activity did not change with time either in the absence or presence of exogenous Stat3 in cells not treated with OSM (not shown).
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Identification of the p21-SBE-binding Proteins--
Nuclear
extracts from cells untreated or treated with OSM for 15 min were
obtained, and electrophoretic mobility shift assays were performed
using p21-SIE1 as a probe. Nuclear extracts from cells treated with OSM
contained a protein(s) that bound the SBE probe and retarded its
mobility (Fig. 7A). This
binding was effectively competed by a 100-fold molar excess of
unlabeled wild type p21-SIE1 oligonucleotide but not by a mutated
p21-SIE1 oligonucleotide. An antibody to Stat3, but not a non-immune
IgG control, reduced the mobility of the protein-DNA complex,
indicating the presence of Stat3. An antibody to Stat1 did not
affect the mobility of the protein-DNA complex; however, it reduced the
intensity of the band.
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The Increases in p21 Protein Are Required for the Pro-differentiating and Anti-apoptotic Effects of gp130 Activation on Osteoblasts-- The relevance of the stimulation of p21 gene expression by IL-6 type cytokines to their biologic effects was investigated using antisense oligodeoxynucleotides to block the synthesis of p21 protein. As shown by the results of the Western blot analysis depicted in Fig. 8A, the OSM-induced increase in p21 protein levels was largely abrogated in cells exposed to antisense oligonucleotides complementary to a region encompassing the initiation codon (AS-IC). Moreover, the AS-IC oligonucleotide blocked the OSM-induced stimulation of AP activity in MG63 cells (Fig. 8B). Two other antisense oligodeoxynucleotides complementary to the middle of the coding region and the 3'end of the p21 gene, respectively, exerted similar inhibitory effects on OSM-induced AP activity. As in the case of the effect of OSM on AP, blocking the expression of the p21 protein with the p21 antisense oligonucleotide AS-IC abolished the protective effect of OSM on Fas-induced apoptosis of MG63 cells (Fig. 8C). A sense oligonucleotide (S-IC), used as a negative control in these experiments, had no significant influence on the effects of OSM.
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DISCUSSION |
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The results of the studies described in this paper demonstrate for
the first time that activation of the gp130 signaling pathway and Stat3
by IL-6 type cytokines leads to the transcriptional activation of the
p21WAF1,CIP1,SDI1 gene in human osteoblastic cells. Moreover,
our findings strongly suggest that stimulation of p21 expression is
required for the pro-differentiation and anti-apoptotic effects of IL-6
type cytokines on this cell type. These observations are in full
agreement with a role of p21 as a downstream effector of the biologic
actions of cytokines that activate STATs, as indicated by evidence that Stat1 and Stat5 mediate -IFN- and thrombopoietin-induced p21 expression, respectively, and that p21 mediates the cell growth arrest
effect of
-IFN on A431 cells and the promotion of differentiation by
thrombopoietin on a megakaryoblastic leukemia cell line (13, 33).
The p21 gene promoter contains three SBEs, designated p21-SIE1, p21-SIE2, and p21-SIE3 (33). The 2.4-kb fragment of the p21 promoter used in our studies, however, contained only p21SIE1, the most proximal of these three elements. Therefore, it is possible that we have underestimated the magnitude of the effect of IL-6 type cytokines on the transcriptional activity of this gene. Nonetheless, Stat3 binds preferentially to p21-SIE1, Stat5 binds to p21-SIE2 and p21-SIE3, but not p21-SIE1, and Stat1 binds to all three (13, 33). In view of this evidence and the fact that Stat3 is the principal STAT activated by IL-6 type cytokines (37), p21-SIE1 is likely to be the most relevant element of the three for the actions of the IL-6 type cytokines.
We have previously reported that IL-6 type cytokines arrest MG63 cell growth at the G1 phase (19). These earlier observations, taken together with the present finding that blockade of p21 prevented the cytokine-induced stimulation of AP activity, are consistent with the contention that acquisition of a specific phenotype is associated with cell cycle withdrawal and that p21 may play a critical role during this process. In support of this, up-regulation of p21 expression, in a p53-independent fashion, and cell cycle arrest have been documented during the differentiation of a number of cell types (9-14, 38, 39). Furthermore, cell cycle arrest in the G1 phase has been shown to facilitate the differentiation process. Thus, overexpression of the Cdk inhibitors p21 and/or p27 caused U937 myelomonocytic cells to differentiate toward a mature monocyte/macrophage phenotype (11). Likewise, overexpression of the Cdk inhibitors p21 and p16 in myoblasts augmented muscle-specific gene expression (40).
In studies not shown here, we have found that besides IL-6 type cytokines, two steroid hormones known to promote osteoblast differentiation in vitro, namely 1,25-dihydroxy-vitamin D3 and dexamethasone, also increase p21 levels in human osteoblast-like MG63 cells. Furthermore, we have observed an increase in the expression of p21 in primary cultures of murine bone marrow cells maintained in the presence of ascorbic acid, another established inducer of osteoblast differentiation. Taken together with the evidence presented in this report, these observations suggest strongly that p21 up-regulation may be a general mechanism of stimulating osteoblastic cell differentiation.
Other Cdk inhibitors that are involved in cell differentiation may act in concert with p21. For example, the greatest increase in differentiation markers of myelomonocytic U397 cells are achieved when p21 and p27 are overexpressed simultaneously (11). In line with this evidence, we have observed that besides up-regulating the expression of p21, gp130 activation also induces smaller increases in the levels of p27 and p15 in MG63 cells (data not shown). These phenomena may account for the fact that OSM-induced increase in AP was not completely suppressed by p21 antisense oligonucleotides.
In addition to the evidence for a role of p21 in cell differentiation, it has been shown previously that muscle cells or tumor cells expressing high levels of p21 are protected against apoptosis (15-17). Furthermore, p53-induced apoptosis in embryonic fibroblasts from a p21-deficient mouse could be prevented by overexpression of p21 (17). The finding of the present paper that blockade of the OSM-induced increase in p21 prevented the anti-apoptotic effect of the cytokine is consistent with these earlier observations.
That IL-6 type cytokines influence osteoblast differentiation was
originally suggested by the observation that mice overexpressing LIF
exhibit sclerotic bone formation (41). Conversely, targeted disruption
of the LIF receptor gene results in decreased bone volume in the
primary spongiosa of developing bone of fetal mice (42). As in the case
of LIF, mice overexpressing OSM exhibit increased bone formation and
enlarged limbs (43). Consistent with these in vivo
observations, we and others have demonstrated that IL-6 type cytokines
induce differentiation of committed osteoblastic cells in
vitro (19, 44-46). In addition, IL-6 plus sIL-6R or LIF stimulate
the differentiation of uncommitted mesenchymal progenitors of the bone
marrow toward the osteoblast phenotype (21), and IL-6 plus sIL-6R or
IL-11 plus soluble IL-11 receptor act on embryonic fibroblasts to
promote commitment toward the osteoblast phenotype, without affecting
the differentiation toward adipocytes or muscle cells (20).
The relevance of p21 regulation by IL-6 type cytokines to osteoblast differentiation and survival in vivo is speculative at this stage. Mice lacking p21WAF1,CIP1,SDI1 appear normal, and histological sections of vertebral bones from these animals were reported to be normal (47). Therefore, p21 is not critical for osteoblast differentiation during development; it may be that other Cdk inhibitors substitute for p21 in these animals. Normal bone development in p21-deficient mice does not rule out, however, a potentially significant role for this gene in states of altered bone cell formation, such as sex steroid deficiency. For example, IL-6-deficient mice also undergo normal bone development. However, after gonadectomy these mice do not develop the increase in osteoblast and osteoclast formation that causes the bone loss observed in their gonadectomized wild type littermates (48, 49). Thus, although IL-6 is not required for normal bone development, it is absolutely required for the bone loss associated with loss of sex steroids. The work presented in this report demonstrates that p21 is a downstream effector of at least some of the IL-6 type cytokines actions on osteoblastic cells. Therefore, it is possible that p21 is also required for the changes in bone cell formation mediated by IL-6 following loss of sex steroids. The elucidation of the role of p21 in states of altered bone cell formation will require similar studies in p21-deficient mice.
In conclusion, the evidence presented here establishes that p21 is a primary response gene for cytokines that activate the gp130/STAT signaling pathway, and a critical effector of their pro-differentiation and anti-apoptotic properties in osteoblastic cells. Further studies will be required to establish whether the transcriptional activation of the p21WAF1,CIP1,SDI1 gene is responsible for the well established differentiation-promoting and anti-apoptotic effects of this family of cytokines on their numerous normal and malignant cell targets.
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ACKNOWLEDGEMENTS |
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We thank Frances Swain and Daniel Felton for technical assistance; Drs. A. Michael Parfitt, Robert L. Jilka, and Charlotte Peterson for critical reading of the manuscript; and the ACRC Office of Grants and Scientific Publications for editorial support.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants R29AR43453 (to T. B.) and PO1AG/AMS13918 (to S. C. M.), by a grant from the Veterans Administration (to S. C. M.), and by American Cancer Society Institutional Grant 187-B (to T. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Div. of Endocrinology
and Metabolism, University of Arkansas for Medical Sciences, 4301 W. Markham, Mail Slot 587, Little Rock, AR 72205. Tel.: 501-686-5130; Fax:
501-686-8148; E-mail: tmbellido{at}life.uams.edu.
The abbreviations used are:
Cdk, cyclin-dependent kinase; IL-6, interleukin-6; IL-11, interleukin-11; LIF, leukemia inhibitory factor; OSM, oncostatin M; gp130, glycoprotein 130; STAT, signal transducer and activator of
transcription; sIL-6R, soluble IL-6 receptor; FBS, fetal bovine serum; AP, alkaline phosphatase; MEM, minimum essential medium; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,
inner salt HRP, horseradish peroxidaseluc, luciferaseLDH, lactate
dehydroxylase-IFN,
-interferonANOVA, analysis of varianceSBE, STAT binding elementdnStat3, dominant negative Stat3kb, kilobase pair(s)CMV, cytomegalovirusPBS, phosphate-buffered
saline.
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REFERENCES |
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