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From the
Received for publication, December 7, 2001
Receptor activator of NF- The osteoclast plays an important role in bone
resorption in vertebrates during development and throughout life
(1). This resorption is counterbalanced by new bone formation from
osteoblasts. This process of coupling the actions of the bone-producing
cells, the osteoblasts, and the bone-resorbing cells, the
osteoclasts, is termed "bone remodeling," a process necessary for
maintaining a constant bone mass throughout the lifetime of vertebrate
organisms. Disruption of this process in humans results in diseases of
bone, including osteoporosis and hypercalcemia of malignancy (2, 3).
Osteoclasts differentiate from cells of the monocyte/macrophage lineage
to become multinuclear, tartrate-resistant acid phosphatase (TRAP)1-positive cells
capable of resorbing bone (4). Osteoclast differentiation is influenced
by hormones and local factors produced by the osteoblasts and stromal
cells (4). One local factor expressed by osteoblasts is receptor
activator of NF- RANKL/RANK signal through tumor necrosis factor receptor-associated
factors to activate multiple signaling pathways thought to be
important for osteoclast differentiation and function, including NF- The microphthalmia-associated transcription factor (MITF) is a basic
helix-loop-helix leucine zipper protein (14, 15) closely related to the
transcription factors TFE3, TFEB, and TFEC (16-18). MITF regulates
osteoclast target genes like TRAP, cathepsin K, and E-cadherin by
binding to a 7-base pair conserved sequence TCANGTG found in the
promoter regions of these genes (19-21). In situ
hybridization experiments confirmed that MITF is expressed in
osteoclasts beginning at the earliest stages of endochondral ossification of long bones (19). A mutant allele of the MITF gene,
mi, encodes a protein product lacking one of four arginines in the basic region of the MITF protein critical for binding to target
genes (14, 22). Osteoclast-like precursor cells derived from mice
homozygous for the mi mutation are incapable of fusing to
form multinuclear cells, lack a ruffled border, express low levels of
TRAP and cathepsin K, and cannot efficiently resorb bone (19, 21, 23,
24). Based on the similarity of the phenotype of the bone
marrow-derived precursor cells blocked for p38 MAPK activity and the
mi/mi osteoclasts-like cells, we hypothesized that MITF may be a target of the p38 MAPK in osteoclasts.
In this study, we investigate whether MITF is a potential target of the
p38 MAPK in osteoclasts. We show that MITF was persistently phosphorylated on a conserved serine 307 in primary osteoclast-like cells in response to RANKL signaling and that phosphorylation of serine
307 correlated with expression of the target gene, TRAP. Further, p38
MAPK could phosphorylate serine 307 in vitro, and phosphorylation of MITF at serine 307 increased the ability of the
factor to stimulate the TRAP promoter activity in transient transfection assays. We conclude that MITF may be a direct target of a
RANKL/p38 signaling pathway that is necessary for osteoclast differentiation and function.
Culture and Analysis of OCLs--
Hematopoietic precursors were
obtained from the bone marrow of wild type mice. OCLs were grown in the
presence of 50 ng/ml colony-stimulating factor-1 (CSF-1) for 3 days.
CSF-1 was lowered to 5 ng/ml, and then RANKL was added at 100 ng/ml for
the indicated times.
SB203580 (Calbiochem) was used at the indicated concentration, 10 µM, and was applied to the cells 30 min before
stimulation with RANKL.
DNA Constructs and Transfections--
For all experiments
presented here, the melanocyte form of the MITF cDNA was used (14,
25). The MITF phosphorylation mutant, termed S307A/P308A, was made by
replacing conserved serine and proline with alanine residues. The point
mutant was verified by sequencing. The full-length form or point
mutants were cloned into a vector providing the influenza hemagglutinin
tag (HA) (Roche Molecular Biochemicals). Wild type TRAP promoter and
the TRAP promoter containing the E box mutation were previously
described (19).
Bacterial expression constructs of His-tagged MKK6b(E) and p38
DNA transfections of RAW 264 cells have been previously described
(19).
Construction of Cell Lines--
RAW 264.7 cells were transfected
with Superfect (Qiagen) following the manufacturer's directions with
either the plasmid expressing HA-tagged S73AMITF or HA-tagged
S73A/S307A/P308AMITF, and cells were selected using 100 µg/ml
Geneticin (Invitrogen). Individual clones were picked and
screened for expression of HA-MITF.
Preparation of Recombinant Proteins--
Recombinant p38
isoforms and MKK6(E) were prepared by a previously described method
(27). MITF corresponding to amino acids 297-377 (nucleotides
1018-1263) was cloned into pGEX2t (Pharmacia) and was purified to 95%
purity by glutathione Sepharose affinity chromatography. Versions of
the protein with either serine or alanine at amino acid position 307 were produced and purified.
RANKL Bacterial Expression and Purification--
RANKL
corresponding to amino acids 158-316 (nucleotides 472-978) was placed
into vector pET 32b (Novagene), purified by native lysis to 95% purity
by nickel-Sepharose (Qiagen) affinity chromatography and eluted in
imidazole. The concentration of soluble RANKL was optimized for
induction of p38 phosphorylation and formation of TRAP-positive
osteoclasts with each preparation.
Immunological Reagents and Analysis--
The peptide
PSTGLSpSPDLVN (where pS represents phosphoserine), corresponding to
amino acids 301-312 of MITF was synthesized (QCB/BIOSOURCE, Hopkinton, MA). The position of
the phosphate at amino acid 307 was confirmed by NMR. The
phosphopeptide was used to immunize two New Zealand White rabbits.
Collected serum was pooled and passed over a column to which the
nonphosphopeptide was coupled, and material that did not bind to this
column was collected and passed over a phosphopeptide affinity column.
Bound material was eluted from this second column with glycine buffer, dialyzed against phosphate-buffered saline, and stored at
Western blotting was performed with nitrocellulose membranes and the
ECL detection system (Kirkegaard and Perry Laboratories). For
Western blots using the antibodies that recognize total or phosphorylated p38, the manufacturer's protocol was followed. For the
phosphospecific MITF antibody, the blot was blocked in TBS (50 mM Tris, pH 7.5, 150 mM NaCl), 0.1%
Tween 20, 5% powdered milk for 2 h at room temperature. The blot
was washed three times in TBS, 0.1% Tween 20. The phosphospecific MITF
antibody was added to fresh blocking buffer and incubated for 2 h
at room temperature. The blot was washed three times in TBS, 0.1%
Tween 20. The secondary antibody was incubated in TBS, 0.1% Tween 20 for 1 h at room temperature. The blot was washed three times in
TBS, 0.1% Tween 20 and developed.
For [35S]methionine labeling experiments, cells were
starved in Dulbecco's modified Eagle's medium lacking methionine. 90 µCi of [35S]methionine (ICN) was added to the cells,
and the cells were labeled for 3 h. Cells were lysed in RIPA (50 mM Tris-HCl, pH 7.6, 125 mM NaCl, 1% Nonidet
P-40, 0.5% deoxycholate) that included 10 µg/ml antipain, 10 µg/ml
leupeptin, 10 µg/ml aprotinin, and 1 mM sodium vanadate.
Cells were incubated in RIPA at 4 °C for 20 min and then centrifuged
at 25,000 rpm for 20 min. Protein G beads (Amersham Biosciences) were
added to the lysate, and the antibody (HA from Babco) was added. The
immunoprecipitates were incubated overnight at 4 °C. The
immunoprecipitates were washed three times in RIPA and run on an 8%
SDS-PAGE.
Kinase Assay--
Full activation of recombinant p38
p38 MAPK activities were measured in an immune complex kinase assay.
RAW264.7 cells were stimulated with RANKL at 100 ng/ml for 15 min.
Cells were lysed in RIPA. Cells were incubated in RIPA at 4 °C for
20 min and then centrifuged at 25,000 rpm for 20 min. The
immunoprecipitates were incubated with anti-p38 antibody (Cell
Signaling Technology) overnight at 4 °C. The immunoprecipitates were
collected and washed three times in RIPA and two times with kinase
buffer (20 mM Mops, pH 7.5, 25 mM Production and Characterization of Antibodies Specific for MITF
Phosphoserine 307--
Osteoclasts that are grown in the presence of
CSF-1 and RANK ligand but have been blocked for the activation of the
p38 MAPK by the specific inhibitor SB203580 remain mononuclear and
stain weakly for the osteoclast marker TRAP (13), a similar phenotype to the osteoclasts that are cultured from the mice homozygous for the
mi mutation (23, 24). This led us to test the hypothesis that MITF might be a target of the p38 MAPK pathway during osteoclast differentiation. We analyzed the amino acid sequence of MITF and discovered a potential p38 phosphorylation site at serine 307 that is
conserved among different species including human, mouse, and chicken
(Fig. 1A). However, the serine
at amino acid position 307 is not conserved among the other related
transcription factors, TFEC, TFE3, and TFEB (29).
To begin to investigate if MITF was phosphorylated at serine 307 in
osteoclasts, an antibody that was specific for the phosphorylated serine at residue 307 was developed (see "Experimental
Procedures"). For this purpose, the peptide PSTGLSpSPDLVN
(corresponding to amino acids 301-312 of MITF) was synthesized and
used to produce polyclonal rabbit serum. Following affinity
purification, the specificity of the antibody for detecting
phosphoserine 307 MITF in Western blotting experiments was tested with
recombinant GST-MITF corresponding to amino acids 297-377. For these
experiments, the GST-MITF region was incubated in vitro with
activated p38
To further characterize the phospho-Ser307-specific
antibody, RAW 264.7 cell lines that stably expressed either HA-tagged
MITF or S307A/P308A HA-tagged MITF were created. The cells were
stimulated with RANKL for various times, and cell extracts were
prepared and immunoprecipitated with the antibody recognizing the HA
epitope. The phosphorylation status of MITF Ser307 was
determined using the phosphospecific MITF antibody (Fig. 1C,
top panel). This analysis demonstrated that
Ser307 was phosphorylated following 15, 30, or 60 min with
RANKL stimulation but that the antibody did not react with the
S307A/P308A MITF protein. The bottom panel of
Fig. 1C represents the blot in the top panel reprobed with
an antibody that recognizes the HA tag to show approximately equal
loading of the HA-containing immunoprecipitates.
MITF Is Rapidly and Persistently Phosphorylated in Osteoclasts
after RANKL Stimulation--
The anti-phospho-MITF antibody was used
to determine the phosphorylation status of endogenous MITF in primary
osteoclast precursors after stimulation by RANKL. Bone marrow-derived
osteoclast precursors were obtained from wild type mice and cultured in
the presence of CSF-1 for 3 days. At this time, RANKL was added to the
bone marrow cell cultures, and nuclear extracts were prepared and
analyzed by Western blotting (Fig. 2).
Using the anti-phospho-Ser307 antibody, MITF
phosphorylation could be detected following 30 min of RANKL stimulation
and persisted following 24 h of continuous RANKL treatment (Fig.
2A, upper panel). When the same blot
was reprobed with a nondiscriminating MITF antibody, approximately equal levels of MITF protein are seen in all lanes (Fig. 2A,
lower panel). MITF appeared as a doublet with
electrophoretic mobility of around 55 and 57 kDa in these experiments,
and both bands are detected by the anti-phospho-Ser307
antibody (Fig. 2A). Previous work in melanocytes has also
indicated that MITF is resolved as a doublet in extracts prepared from
this cell type and that the upper band is due to c-Kit-mediated
phosphorylation of conserved serine residue 73 (30). Ser73
is also phosphorylated in osteoclasts in response to CSF-1, accounting for the doublet band pattern in this cell line as well (31). As shown
below (see Fig. 5) mutation of Ser73 to alanine results in
a loss of the slower migrating band (see Fig. 5).
Real time PCR was used to measure expression of the MITF target gene
TRAP (19) following RANKL stimulation of osteoclasts (Fig.
2B). These experiments demonstrated that TRAP expression was
increased ~7-fold following 30 min of RANKL treatment and remained
elevated 7-fold, following 24 h of RANKL treatment, in good
agreement with the observed increase in MITF Ser307 phosphorylation.
As previously reported in RAW 264.7 cells (13), we were able to detect
the phosphorylated, activated form of p38 MAPK in primary osteoclasts
following stimulation with RANKL (Fig. 2C). The
phosphorylation of p38 MAPK, like the phosphorylation of MITF, was
persistent, since we were able to detect phosphorylated p38 MAPK even
24 h after stimulation with RANKL (Fig. 2C,
top panel). This blot was stripped and
reprobed with an antibody that recognizes all forms of p38 to
ensure that there was approximately equal loading of protein in all
lanes (Fig. 2C, bottom panel). This second antibody detected the same 42-kDa protein in all samples, even
the sample that received only CSF-1.
The effects of the specific p38 MAPK inhibitor, SB203580, on MITF
Ser307 phosphorylation and TRAP gene expression were
examined (Fig. 2, A and B). This analysis
demonstrated that MITF Ser307 phosphorylation in response
to RANKL could be inhibited by SB203580 pretreatment of osteoclasts
(Fig. 2A, lane 3). Additionally, TRAP gene induction by RANKL could be reduced from 7- to 3-fold by SB203580
pretreatment (Fig. 2B).
p38 Can Phosphorylate MITF in Vitro--
To provide additional
evidence linking MITF phosphorylation to p38 MAPK, in vitro
kinase reactions were performed. Two types of assays were used. First,
p38 MAPK immunoprecipitated from RAW264.7 was used in immune kinase
reactions with GST-MITF substrate (Fig. 3A). The immune kinase
experiments demonstrated that the p38 immunoprecipitate prepared from
RANKL-treated cells phosphorylated the GST-MITF protein, whereas one
prepared from unstimulated cells did not (Fig. 3A, compare
lane 1 and lane 3).
Additionally, an MITF protein containing S307A/P308A mutations was a
poor substrate in the assay, whether p38 was immunoprecipitated from
RANKL-treated or -untreated cells (Fig. 3A, lanes
2 and 4).
In the second assay, the ability of recombinant p38
c-Jun N-terminal kinase MAP kinase could not phosphorylate GST-MITF
substrates in vitro in either type of assay, under
conditions where a GST-Jun substrate could be phosphorylated (data not shown).
Dominant Active Forms of Rac1 and MKK6 Can Collaborate with MITF to
Superactivate the TRAP Promoter in Transient Assays--
Rac1 and has
been previously shown to be able to activate the p38 MAPK cascade (28).
Further, MKK6 has been previously shown to be a MAPK kinase that is a
specific activator of p38 MAPKs (28, 32). To provide additional
evidence documenting MITF as a downstream target of the RANKL/p38 MAPK
signaling pathway, we tested whether MITF's ability to transactivate
the TRAP promoter fused to a firefly luciferase reporter gene was
stimulated by Rac1(15L) or MKK6(E), constitutive active forms of these
molecules. Experiments were performed in the macrophage/osteoclast cell
line RAW 264.7 (Fig. 4).
In these experiments, co-transfection of the TRAP reporter with MITF
expression vector resulted in approximately a 10-fold increase in
promoter activity compared with control basal activity in agreement
with our previously published results (19) (Fig. 4). When either
Rac1(15L) or MMK6(E) expression vectors were co-transfected with the
TRAP reporter, there was an ~4-5-fold increase in TRAP promoter
activity compared with basal activity (Fig. 4). When the combination of
Rac1(15L) and MITF expression vectors was co-transfected with the TRAP
promoter, there was a more than 100-fold increase in promoter activity
relative to the control (Fig. 4, compare bar 1 with bar 5), an effect that was more than
additive of the effects seen with MITF or Rac1(15L) expression vectors
alone. When a TRAP reporter that contains point mutations in the E
box-related MITF-binding site identified in our previous studies (19)
was tested in combination with MITF and Rac1(15L), activation was abolished (Fig. 4, compare bar 5 with
bar 6).
Co-transfection of MITF and MKK6(E) with either TRAP reporter produced
similar results, a 100-fold activation of wild type reporter and no
activation of the MITF-binding site mutation (Fig. 4, compare bar
5 with bar 6 and compare bar
7 with bar 8, respectively). These
experiments indicate that MITF collaborates with the Rac/MKK6 signaling
pathway to stimulate TRAP promoter activity dependent on the cis-acting
MITF-binding site present in the TRAP promoter.
Serine 307 and Proline 308 Are Necessary for MITF
Collaboration with Rac1 and MKK6--
As discussed above, serine 307 is phosphorylated in vivo in response to RANKL signaling and
is a substrate for p38 MAPK in vitro. To test the role of
this phosphorylation site in MITF collaboration with either Rac1(15L)
or MKK6(E), the HA-MITF protein containing S307A/P308A (see Fig. 1) was
studied in the transient transfection assays in RAW264.7 cells (Fig.
5A). When co-transfected with
the TRAP reporter, the MITF S307A/P308A expression vector was able to
transactivate the TRAP promoter to similar levels as observed with the
wild type MITF gene (Fig. 5A). In contrast, MITF S307A/P308A co-transfected with constitutively active Rac1 or MKK6 could not activate the TRAP promoter to the same levels as wild type MITF (Fig.
5A). Mutation of other potential serine phosphorylation sites (e.g. an S73A point mutation in the previously
characterized Erk phosphorylation site (30, 31)) had no affect on
stimulation of MITF activity by either Rac1(15L) or MKK6(E)
(data not shown).
To determine whether the alanine substitutions affected protein
stability, we examined expression of HA-tagged MITF proteins following
transfection of COS cells (Fig. 5B). In these experiments, both MITF wild type and S307A/P308A forms were again resolved as
doublets. However, MITF with the S73A substitution ran as a single,
faster migrating band, consistent with the previous finding that
phosphorylation of this site by the Erk MAPK pathway results in the
band with lower electrophoretic mobility (30, 31). No significant
difference in the levels of the wild type MITF or MITF S307A/P308A
proteins could be detected in this assay (Fig. 5B). Taken
with the data above showing that S307A/P308A MITF can be stably
expressed to the same extent as wild type protein in RAW264.7 cells
(Fig. 1C, bottom panel), these results
indicate that MITF protein stability is not grossly affected by these
point mutations.
p38
To test if dominant negative versions of the four isoforms of p38 (27)
could inhibit Rac1 activation of MITF, transient transfections were
performed with RAW 264.7 cells (Fig. 6B). As shown above,
co-transfection of MITF and Rac1 (15L) expression vectors with the TRAP
reporter resulted in 100-fold activation of the TRAP promoter relative
to the control (Fig. 6B, compare bar 1 with bar 4). When dominant negative expression
vectors for p38 In this study, we present evidence indicating that MITF is a
direct target of a RANKL/p38 signaling pathway and that activation of
MITF by this pathway is necessary for efficient expression of TRAP.
MITF is thus defined as a nuclear target for RANKL signaling that,
along with p38 MAPK activity, is persistently, and not transiently, activated by RANKL. Studies on other signaling pathways activated by
RANKL, specifically the I The p38 kinases constitute a distinct MAPK subfamily that plays a role
in adaption, homeostasis, and stress responses (39). In addition, the
p38 pathway has been implicated in the induction of myoblast
differentiation (40, 41). There are interesting parallels between the
role of p38 in muscle differentiation and in osteoclast
differentiation. The myogenic MEF transcription factors are targets of
p38 signaling (27, 42). Additionally, the p38 MAPK pathway is
persistently activated and maintained during the whole process
of myotube formation (41). As is the case for the p42/p44 MAPK kinase
in neuronal cell differentiation (43), persistent activation of
signaling appears necessary for the ability of the p38 MAPK pathway to
promote cell differentiation in myoblasts and osteoclast precursors. In
muscle differentiation, the identity of the factor that triggers p38
activation is unknown (41), unlike osteoclast differentiation, where
RANKL is the factor that stimulates differentiation. The RANKL/p38
MAPK/MITF pathway provides a paradigm to define how persistent
activation of signaling occurs.
MITF regulates distinct sets of target genes in different cell types,
and a major question is what mechanisms allow for cell type-specific
actions of MITF. The results presented here suggest that cell
type-specific signaling events may contribute to the ability of MITF to
selectively alter the expression of target genes in one cell type
versus another. For example, the expression of RANK in
osteoclast precursors distinguishes this cell type from melanocytes and
thus allows for cell type-specific phosphorylation and activation of
MITF. Importantly, the conserved Ser307 residue is not
conserved among the related family members TFE3, TFEC, and TFEB (29).
This is distinct from the conserved Ser73 residue, a target
for the Raf/p42/p44 MAPK pathway (30, 31), a site conserved
among the entire family (29). Thus, MITF may be regulated by the
p42/p44 MAPK pathway in all cell types in which it is expressed, but
phosphorylation of serine 307 may represent one mechanism of regulating
MITF activity that is unique to osteoclasts.
How does phosphorylation at Ser307 modulate MITF activity
in osteoclasts? Recent results suggest that two probable mechanisms should be considered. First, our laboratory has recently demonstrated that MITF and the Ets family member PU.1 interact uniquely in osteoclasts to regulate target genes and to promote osteoclast differentiation (44). One hypothesis is that phosphorylation of MITF by
the p38 MAPK pathway increases the affinity of the interaction between
MITF and PU.1 and thus increases the ability of this pair of
transcription factors to stimulate osteoclast differentiation. A second
hypothesis is that phosphorylation of MITF lowers its affinity for a
co-repressor. For example, in muscle differentiation,
calcium/calmodulin signaling leads to a dissolution of a complex
between MEF2 proteins and histone deacetylases and allows MEF2 to
interact with the helix-loop-helix factor MyoD to trigger muscle
differentiation (45). The two hypotheses are not mutually exclusive. It
could be that phosphorylation of MITF on serine 307 leads to a lower
affinity for a co-repressor and subsequent high affinity interaction
with PU.1. Further analysis will be required to determine whether these
hypotheses can account for the ability of p38 MAPK to activate
MITF.
The work presented here indicates that osteoclast-specific signaling
events may contribute to the ability of MITF to regulate osteoclast
gene expression and differentiation. These results suggest a unique set
of molecular targets that might allow modulation of osteoclast
differentiation and activity in bone disorders caused by the uncoupling
of osteoblast and osteoclast action (e.g. osteoporosis).
We acknowledge the role of the Keck Genetic
facility in maintaining the mouse colony used in this work and Lori
Nelsen for expert technical assistance.
*
This work was supported by NIAMS, National Institutes of
Health (NIH), Grant AR-44719 (to M. C. O.).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.
§
Supported by NIH National Research Service Award F32-AR08568.
Published, JBC Papers in Press, January 15, 2002, DOI 10.1074/jbc.M111696200
The abbreviations used are:
TRAP, tartrate-resistant acid phosphatase;
RANK, receptor activator of
NF-
Microphthalmia Transcription Factor Is a Target of the
p38 MAPK Pathway in Response to Receptor Activator of NF-
B
Ligand Signaling*
§,,
Department of Molecular Genetics and the
Comprehensive Cancer Center, Ohio State University, Columbus, Ohio
43210 and the ¶ Department of Immunology, The Scripps Research
Institute, La Jolla, California 92037
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B ligand (RANKL)
activates signaling pathways that regulate osteoclast differentiation,
function, and survival. The microphthalmia transcription factor (MITF)
is required for terminal differentiation of osteoclasts. To determine whether MITF could be a target of RANKL signaling, a phosphospecific MITF antibody directed against conserved residue
Ser307, a potential mitogen-activated protein kinase
(MAPK) site, was produced. Using this antibody, we could demonstrate
that MITF was rapidly and persistently phosphorylated upon stimulation
of primary osteoclasts with RANKL and that phosphorylation of
Ser307 correlated with expression of the target gene
tartrate-resistant acid phosphatase. MITF phosphorylation at
Ser307 also correlated with persistent activation of p38
MAPK, and p38 MAPK could utilize MITF Ser307 as a substrate
in vitro. The phosphorylation of MITF and activation of
target gene expression in osteoclasts were blocked by p38 MAPK inhibitor SB203580. In transient transfections, a constitutively active
Rac1 or MKK6 gene could collaborate with MITF to activate the
tartrate-resistant acid phosphatase gene promoter dependent on
Ser307. Dominant negative p38 
and 
could inhibit the
collaboration between upstream signaling components and MITF in the
transient assays. These results indicate that MITF is a target for the
RANKL signaling pathway in osteoclasts and that phosphorylation of MITF leads to an increase in osteoclast-specific gene expression.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B ligand (RANKL) (5-10). RANKL is a member of the
tumor necrosis factor superfamily that is important for maturation of
T- and B-cells as well as osteoclasts (7). Mice containing a targeted
deletion in either the RANK receptor or RANKL lack differentiated
osteoclasts and develop severe osteopetrosis (7-9).
B, mitogen-activated protein kinase (MAPK) pathways, Src kinase, and phosphatidylinositol 3-kinase pathways (11, 12). Recently, it has
been shown that inhibition of the p38 signaling pathway in bone
marrow-derived osteoclast precursor cells by treatment with the drug
SB203580 inhibited the formation of multinuclear, functional
osteoclasts that expressed TRAP in response to RANKL treatment (13). In
contrast, the drug PD98059, a specific inhibitor of the MAPK p42/44
pathway, had no effect on the differentiation of bone marrow cells.
These results indicate that the p38 MAPK signaling pathway is involved
in RANKL-induced differentiation of bone marrow-derived precursor cells
(13).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(26),
dominant negative expression plasmids for p38 , , 
, and 
isoforms (27), and MKK6(E) expression plasmid were all previously
described (28).
70 °C.
Polyclonal phosphorylation-independent MITF antibody has been described
previously (20). p38 - and -specific antibodies were previously
described (26). Antibodies that recognized p38 MAPK were obtained from
New England Biolabs Cell Signaling.
in vitro was achieved by incubation with recombinant MKK6(E)
at a 5:1 molar ratio at 37 °C for 15 min in the presence of ATP as
previously described (27). In vitro kinase assays were
carried out at 37 °C for 15 min, using 0.2 µg of recombinant
kinase, 5 µg of GST-MITF, 250 µM ATP, and 12 µCi of
[-32P]ATP in 20 µl of kinase reaction buffer.
Reactions were terminated by the addition of SDS sample buffer.
Reaction products were resolved on a 10% SDS-PAGE. Phosphorylated
proteins were visualized by autoradiography.
-glycerol
phosphate, 10 mM MgCl2, 1 mM
dithiothreitol, 1 mM sodium vanadate). Immunoprecipitates of anti-p38 were mixed with 0.5 µg of GST-MITF, 150 µM
ATP, and 12 µCi of [-32P]ATP in 50 µl of kinase
buffer. The reactions were further incubated at 30 °C for 30 min.
Reactions were terminated by the addition of SDS sample buffer.
Reaction products were resolved on a 10% SDS-PAGE. Phosphorylated
proteins were visualized by autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (25K):
[in a new window]
Fig. 1.
Generation of phosphospecific MITF
antibody. A, alignment of potential p38 MAPK
phosphorylation sites in MITF. The sequence displayed is from amino
acid 299 to 320. The conserved residues important for p38 MAPK
phosphorylation are indicated in large boldface
letters. B, phosphospecific MITF antibody
recognizes only recombinant phosphorylated MITF. GST-MITF (amino acids
297-377) was incubated with nonradioactive ATP in the presence
(lanes 1 and 3) or the absence
(lanes 2 and 4) of His-tagged p38 .
The top panel is Western blot using
phosphospecific MITF antibody. Lanes 1 and
2, wild type GST-MITF; lanes 3 and
4, S307A/P308A GST-MITF. Bottom panel,
blot in the top panel reprobed with an antibody
recognizing GST. C, phosphospecific MITF antibody recognizes
HA-MITF stably expressed in RAW 264.7 cells upon stimulation with
RANKL. Immunoprecipitates with HA antibody were run on an 8% SDS-PAGE.
The top panel is a Western blot probing with the
phosphospecific MITF antibody. The left half of
the panel contains HA immunoprecipitates from cells that
stably express HA-MITF containing a replacement of the serine 307 and
proline 308 to alanines, whereas the right half
of the panel contains HA immunoprecipitates from cells
expressing HA-MITF with no change at serine 307 and proline 308. The
bottom panel is the blot in the top
panel reprobed with an antibody that recognizes the HA
tag.
and ATP (Fig. 1B). These experiments
showed that only GST-MITF that had been incubated with the p38
preparation was recognized by the antibody (Fig. 1B,
lane 1 versus lane
2). Recombinant protein containing alanine substituted for
serine at amino acid position 307 was not recognized by the
phosphospecific antibody in either the presence or absence of activated
p38 MAPK. (Fig. 1B, compare lanes 3 and 4 with lane 1). When the blot in
Fig. 1B was stripped and reprobed with an antibody specific
for the GST moiety, we were able to detect the recombinant protein in
all four lanes (Fig. 1B, bottom
panel).
View larger version (33K):
[in a new window]
Fig. 2.
RANKL stimulates phosphorylation of MITF at
serine 307. A, phosphospecific MITF antibody that
recognizes phosphorylated MITF after RANKL stimulation of osteoclasts.
The top panel is a Western blot of nuclear
extracts from osteoclasts run on an 8% SDS-PAGE and probed using
phosphospecific MITF antibody. The numbers above
the lanes indicate the hours after RANKL stimulation that
the extracts were collected. The bottom panel is
the same blot in the top panel reprobed with an
antibody that recognizes all forms of MITF. B, real time
quantitative RT-PCR (Taqman assay) was performed using RNA from bone
marrow cultures treated for the same time with RANKL as indicated in
A. Both TRAP and glyceraldehyde-3-phosphate dehydrogenase
RNA were quantitated. All values in the chart are expressed as the
ratio of TRAP to glyceraldehyde-3-phosphate dehydrogenase expression
and then further normalized to the CSF-1-only control. C,
p38 MAPK is phosphorylated in response to RANKL stimulation in
osteoclasts. The top panel is a Western blot of
whole cell extracts from osteoclasts, run on a 10% SDS-PAGE and probed
using phosphospecific p38 antibody. The numbers
above the lanes indicate the hours after RANKL
stimulation that the extracts were collected. The bottom
panel is the same blot in the top
panel reprobed with an antibody that recognizes all forms of
p38 MAPK.
View larger version (23K):
[in a new window]
Fig. 3.
p38 can phosphorylate MITF in
vitro. A, top panel,
RAW 264.7 cell extracts stimulated (lanes 1 and
2) with RANKL for 15 min or unstimulated (lanes
3 and 4). p38 was immunoprecipitated and
incubated with GST-MITF (amino acids 297-377) and
[ -32P]ATP. Lanes 1 and
3, wild type GST-MITF; lanes 2 and
4, S307A/P308A GST-MITF. The bottom
panel shows a p38 Western blot. B, recombinant
p38 can phosphorylate GST-MITF (amino acids 297-377). Recombinant
GST-MITF was incubated with [-32P]ATP and purified,
activated His-tagged p38 for 15 min and run on a 10% SDS-PAGE. In
the top panels, lanes 1,
3, and 5 contain activated p38 , and
lanes 2, 4, and 6 have no
activated p38 . Lanes 1 and 2 contain wild type GST-MITF as the substrate, lanes
3 and 4 contain S307A/P308A GST-MITF as the
substrate, and lanes 5 and 6 contain
GST as a substrate. The bottom panel shows a GST
Western blot.
to phosphorylate
GST-MITF was tested. Recombinant p38, activated in vitro
by constitutively active MKK6 (28), could phosphorylate wild type
GST-MITF (Fig. 3B, lane 1). The level
of phosphorylation of the Ser307 protein was ~5-fold
higher than phosphorylation of S307A-mutated MITF protein (Fig.
3B, compare lanes 1 and 3).
The phosphorylation of GST-MITF depended on the addition of both MKK6
and p38 (Fig. 2C, lanes 2 and
4), and GST alone was not phosphorylated by either kinase
(Fig. 2C, lanes 5 and 6).
In similar experiments, the p38 isoform could also phosphorylate
GST-MITF, but the p38 isoform could not (data not shown).
View larger version (20K):
[in a new window]
Fig. 4.
Constitutively active Rac1 and MKK6
collaborate with MITF to superactivate the TRAP promoter. Activity
of TRAP-luciferase promoters in RAW 264.7 cells is shown. The activity
of MITF alone and MITF and Rac1(15L) or MKK6(E) were compared on a wild
type TRAP promoter and a TRAP promoter containing a mutation in the E
box. In each experiment, 2.5 µg of TRAP promoter (wild type or E
box-mutated) was co-transfected with 0.5 µg of MITF expression vector
(or empty expression vector for basal activity) or with 0.4 µg of
Rac1 or MKK6 expression vector alone or together as indicated. Activity
is expressed as relative luciferase activity. The average of three
independent experiments performed in duplicate is shown, and
error bars indicate S.D.
View larger version (20K):
[in a new window]
Fig. 5.
MITF Ser307 is necessary for
collaboration with Rac1 or MKK6. A, activity of point
mutants measured in transient transfection assays in RAW 264.7 cells.
TRAP luciferase reporter construct (2.5 µg) was co-transfected with
0.5 µg of expression vector for either wild type MITF, MITF
containing the indicated point mutant, or 0.4 µg of Rac1 15L or
MKK6(E) alone or together. Activity is expressed as relative luciferase
activity. The averages of three independent experiments performed in
duplicate are shown, and the error bars indicate
S.D. B, MITF proteins were expressed transiently in COS
cells. Immunoprecipitation of 35S-labeled cell extracts
with an HA-specific antibody is shown. Lane 1, HA
vector; lane 2, HA-tagged wild type MITF;
lane 3, HA-tagged MITF S307A/P308A;
lane 4, HA-tagged MITF S73A/P74A (the Erk1 site)
(30, 31). The immunoprecipitates were run on an 8% SDS-PAGE. The
arrows indicate the positions of the wild type or mutated
MITF protein. The specific MITF bands present with lower
electrophoretic mobility probably represent the protein phosphorylated
at serine 73 (see "Results" for details) (30, 31).
and Dominant Negative Can Inhibit Rac1/MITF
Activation--
There are four p38 isoforms (, , , and ),
encoded by separate genes (26, 33-35). We were interested in
determining which isoform(s) of p38 was involved in osteoclastogenesis.
Since SB203580 has been shown to inhibit only p38 and isoforms
at low doses used in the experiments presented above (36), it
was likely that one or both of these isoforms were involved in
osteoclast differentiation. Western analysis was performed with p38
antibodies specific for the p38 or isoforms (26). The p38 isoform was detected in both RAW 264.7 and OCL whole cell extracts
(Fig. 6A, lanes
1 and 2). However, we were unable to detect
expression of p38 in either RAW 264.7 or OCLs, although the
antibody did recognize recombinant p38 protein included on the blot
as a positive control (Fig. 6A, compare lanes
5, 6, and 7, respectively).
View larger version (22K):
[in a new window]
Fig. 6.
p38 dominant negatives inhibit Rac1
superactivation of MITF. A, Western blot using p38 and -specific antibodies. Lanes 1 and
5, RAW whole cell extracts; lanes 2 and 6, OCL whole cell extracts; lanes
3 and 7, recombinant His-tagged p38 ;
lanes 4 and 8, recombinant His-tagged
p38 . The arrows indicate the positions of recombinant or
p38 MAPK found in the whole cell extracts. The extracts were run on a
10% SDS-PAGE. The left top blot was
probed with p38 -specific antibody; the right
top blot was probed with p38 -specific
antibody; and the left and right
bottom blots were probed with p38 antibody that
recognizes all isoforms of p38. B, activity of MITF
co-transfected with p38 dominant negatives measured in transient
transfections in RAW 264.7 cells. TRAP luciferase reporter construct
(2.5 µg) was co-transfected with 0.5 µg of MITF expression vector,
0.4 µg of Rac1 15L (also contains empty p38 expression vector) or
MITF, Rac1 15L, and 1 µg of p38 dominant negatives ( and in
left panel, and in right
panel, respectively). Activity is expressed as relative
luciferase activity. The average of three independent experiments
performed in duplicate is shown, and the error
bars indicate S.D.
and were co-transfected with MITF and Rac1
expression vectors, we detected no significant decrease in luciferase
activity compared with when MITF and Rac1 expression vectors were
co-transfected together (Fig. 6B, right
panel). However, when expression vectors for the isoforms
p38 and were co-transfected with MITF and Rac1 expression
vectors, the luciferase activity of the TRAP promoter was similar to
levels seen when only MITF or Rac1 was co-transfected with the
TRAP-luciferase reporter (Fig. 6B, left
panel, compare bar 4 with either
bar 5 or bar 6,
respectively). Although our analysis indicated the p38 isoform was
not expressed in primary osteoclast-like cells, the fact that the
dominant-negative version of the gene could still interfere with Rac1
activation of MITF is not surprising, given the high level of
similarity between the p38 and isoforms.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B/NF-B and c-Jun N-terminal kinase/c-Jun pathways, have not addressed the issue of persistence but have only
studied activation within 30 min of RANKL treatment (10, 37, 38). RANKL
can stimulate transient, acute inflammatory responses as well as
promoting osteoclast differentiation (7). Thus, defining whether
signaling pathways are activated in a transient or persistent manner is
probably critical for understanding how RANKL promotes inflammatory
responses as opposed to differentiation.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Dept.
of Molecular Genetics, Ohio State University, 496 W. 12th Ave.,
Columbus, OH 43210. Tel.: 614-688-3824; Fax: 614-688-8727; E-mail:
ostrowski.4@osu.edu.
ABBREVIATIONS
B;
RANKL, RANK ligand;
MAPK, mitogen-activated protein kinase;
MITF, microphthalmia transcription factor;
CSF, colony-stimulating
factor;
HA, hemagglutinin;
RIPA, radioimmune precipitation assay;
Mops, 4-morpholinepropanesulfonic acid;
GST, glutathione
S-transferase;
OCL, osteoclast.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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D. Xu, S. Wang, W. Liu, J. Liu, and X. Feng A Novel Receptor Activator of NF-{kappa}B (RANK) Cytoplasmic Motif Plays an Essential Role in Osteoclastogenesis by Committing Macrophages to the Osteoclast Lineage J. Biol. Chem., February 24, 2006; 281(8): 4678 - 4690. [Abstract] [Full Text] [PDF]
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 C. Goding and F. L. Meyskens Jr. Microphthalmic-Associated Transcription Factor Integrates Melanocyte Biology and Melanoma Progression Clin. Cancer Res., February 15, 2006; 12(4): 1069 - 1073. [Full Text] [PDF]
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D. M. Molina, S. Grewal, and L. Bardwell Characterization of an ERK-binding Domain in Microphthalmia-associated Transcription Factor and Differential Inhibition of ERK2-mediated Substrate Phosphorylation J. Biol. Chem., December 23, 2005; 280(51): 42051 - 42060. [Abstract] [Full Text] [PDF]
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K. Kim, J. H. Kim, J. Lee, H.-M. Jin, S.-H. Lee, D. E. Fisher, H. Kook, K. K. Kim, Y. Choi, and N. Kim Nuclear Factor of Activated T Cells c1 Induces Osteoclast-associated Receptor Gene Expression during Tumor Necrosis Factor-related Activation-induced Cytokine-mediated Osteoclastogenesis J. Biol. Chem., October 21, 2005; 280(42): 35209 - 35216. [Abstract] [Full Text] [PDF]
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A. J. Miller, C. Levy, I. J. Davis, E. Razin, and D. E. Fisher Sumoylation of MITF and Its Related Family Members TFE3 and TFEB J. Biol. Chem., January 7, 2005; 280(1): 146 - 155. [Abstract] [Full Text] [PDF]
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W. Liu, D. Xu, H. Yang, H. Xu, Z. Shi, X. Cao, S. Takeshita, J. Liu, M. Teale, and X. Feng Functional Identification of Three Receptor Activator of NF-{kappa}B Cytoplasmic Motifs Mediating Osteoclast Differentiation and Function J. Biol. Chem., December 24, 2004; 279(52): 54759 - 54769. [Abstract] [Full Text] [PDF]
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S. Corre, A. Primot, E. Sviderskaya, D. C. Bennett, S. Vaulont, C. R. Goding, and M.-D. Galibert UV-induced Expression of Key Component of the Tanning Process, the POMC and MC1R Genes, Is Dependent on the p-38-activated Upstream Stimulating Factor-1 (USF-1) J. Biol. Chem., December 3, 2004; 279(49): 51226 - 51233. [Abstract] [Full Text] [PDF]
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M. Matsumoto, M. Kogawa, S. Wada, H. Takayanagi, M. Tsujimoto, S. Katayama, K. Hisatake, and Y. Nogi Essential Role of p38 Mitogen-activated Protein Kinase in Cathepsin K Gene Expression during Osteoclastogenesis through Association of NFATc1 and PU.1 J. Biol. Chem., October 29, 2004; 279(44): 45969 - 45979. [Abstract] [Full Text] [PDF]
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H. So, J. Rho, D. Jeong, R. Park, D. E. Fisher, M. C. Ostrowski, Y. Choi, and N. Kim Microphthalmia Transcription Factor and PU.1 Synergistically Induce the Leukocyte Receptor Osteoclast-associated Receptor Gene Expression J. Biol. Chem., June 20, 2003; 278(26): 24209 - 24216. [Abstract] [Full Text] [PDF]
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Y. Liu, Z. Shi, A. Silveira, J. Liu, M. Sawadogo, H. Yang, and X. Feng Involvement of Upstream Stimulatory Factors 1 and 2 in RANKL-induced Transcription of Tartrate-resistant Acid Phosphatase Gene during Osteoclast Differentiation J. Biol. Chem., May 30, 2003; 278(23): 20603 - 20611. [Abstract] [Full Text] [PDF]
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P.-W. Tsai, S.-G. Shiah, M.-T. Lin, C.-W. Wu, and M.-L. Kuo Up-regulation of Vascular Endothelial Growth Factor C in Breast Cancer Cells by Heregulin-beta 1. A CRITICAL ROLE OF p38/NUCLEAR FACTOR-kappa B SIGNALING PATHWAY J. Biol. Chem., February 14, 2003; 278(8): 5750 - 5759. [Abstract] [Full Text] [PDF]
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