1. Volume 8, Issue 4, Pages 749-758 (October 2001)
Linkage of M-CSF Signaling to Mitf, TFE3, and the Osteoclast Defect in Mitfmi/mi Mice 
Katherine N. Weilbaecher, Gabriela Motyckova, Wade E. Huber, Clifford M. Takemoto, Timothy J. Hemesath, Ying Xu, Christine L. Hershey, Nikki R. Dowland, Audrey G. Wells, David E. Fisher 
Molecular Cell 
Volume 8, Issue 4, Pages (October 2001)
DOI: /S (01)

2. Figure 1
M-CSF and ERK1/2 Activation Enhance Osteoclast Maturation and Fusion
(A) Mouse wild-type and op/op macrophages cultured in the presence of M-CSF and RANKL differentiate into TRAP-positive osteoclasts (bottom panels) capable of calcium phosphate resorption (top panels). M-CSF deprivation prevents formation of calcium phosphate resorbing cells or multinucleated TRAP-positive cells.
(B) Wild-type bone marrow-derived macrophages were cultured in the presence of RANKL and M-CSF where indicated. The addition of an increasing amount of MEK inhibitor (20 μM, 40 μM) produces a dose-dependent decrease in the formation of multinucleated (3+ nuclei) TRAP-positive cells (Day 4). Doses of MEK inhibitor up to 40 μM did not measurably affect viability (<1% trypan blue positive cells, data not shown).
(C) MEK inhibitor treatment (40 μM) does not affect viability of bone marrow derived macrophages cultured in the presence of RANKL and M-CSF, as indicated, and proportionally increases the number of immature, mononuclear TRAP-positive cells
Molecular Cell 2001 8, DOI: ( /S (01) )

3. Figure 2
M-CSF Induces ERK/MAPK Activation and Mobility Shift of Mitf and TFE3 in Osteoclasts
(A) Human osteoclasts were starved and stimulated with hM-CSF. M-CSF induced an upward mobility shift of the lower Mitf bands (4 arrows). The 50 kDa immunoglobulin heavy chain (IgH) band (single arrow) represents internalization of human M-CSF-neutralizing antibodies added during the starvation period.
(B) A similar mobility shift of TFE3 occurred after incubation of mouse osteoclasts with M-CSF (2 arrows). The murine M-CSF-neutralizing antibody is not internalized.
(C) M-CSF induces ERK1/2 phosphorylation. Incubation of M-CSF-stimulated human osteoclasts with increasing concentrations of MEK inhibitor for 1 hr (lane 2, 30 μM; lane 3, 60 μM; lane 4, 90 μM) suppresses MAPK phosphorylation and the Mitf mobility shift (2 arrows). MAPK blotting serves as a loading control.
(D) MEK inhibitor suppressed the M-CSF-induced TFE3 mobility shift (see arrow) in mouse osteoclasts (MEK inhibitor concentrations: lane 3, 30 μM; lane 4, 60 μM; lane 5, 90 μM)
Molecular Cell 2001 8, DOI: ( /S (01) )

4. Figure 3
M-CSF Induced Phosphorylation of Mitf and TFE3 at the Conserved PXSP Motif
(A) A MAPK phospho-acceptor site is conserved between Mitf, TFE3, TFEB, and TFEC (Hemesath et al., 1998; Rehli et al., 1999; Roman et al., 1992). The 13 AA phosphorylated peptide used to generate the phospho S-73-specific Mitf antibody (αP-73) is underlined.
(B) Mitf protein was immunoprecipitated (IP) from TPA-stimulated and unstimulated 501-mel melanoma cells and then immunoblotted (Western) with α-Mitf. The arrow indicates the serine 73 phosphorylated Mitf isoform previously characterized by 2D phosphotryptic mapping in melanoma cells (Hemesath et al., 1998).
(C) Plasmids encoding wild-type melanocyte Mitf and serine 73-to-alanine Mitf mutant forms were transfected into the Cos-7 cell line. Untransfected Cos-7 cells do not express Mitf (data not shown). αP-73 antibody recognizes only the serine 73 phosphorylated upper Mitf band, but not the S73A mutant.
(D) αP-73 immunoprecipitates only the 2 upper mobility Mitf isoforms in M-CSF-stimulated osteoclasts, but not in the presence of MEK inhibitor.
(E) αP-73 immunoprecipitates the 2 upper TFE3 bands from M-CSF-stimulated osteoclasts, but not from the MEK inhibitor treated cells
Molecular Cell 2001 8, DOI: ( /S (01) )

5. Figure 4
M-CSF-Mediated Phosphorylation of Mitf and TFE3 Triggers Recruitment of the Transcriptional Coactivator p300 within Osteoclasts and Does Not Affect DNA Binding of Mitf
(A) Wild-type (WT), Mitfmi/mi, and op/op osteoclasts were generated from spleen cells using M-CSF and RANKL. M-CSF induces phosphorylation of Mitf and TFE3 in osteoclasts from all three mouse strains. Beta tubulin serves as a loading control, and Phospho-ERK/MAPK probe indicates M-CSF activation.
(B) Electrophoretic mobility shift assay. M-CSF-induced phosphorylation of Mitf does not affect Mitf's DNA binding ability. The DNA binding ability of Mitf from nuclear extracts of day 8 M-CSF-stimulated, wild-type osteoclasts, M-CSF-stimulated cells in the presence of MEK inhibitor, or unstimulated osteoclasts was tested on a probe derived from the human cathepsin K promoter spanning an E-box element previously shown to bind Mitf and to be important in the regulation of cathepsin K expression by Mitf and TFE3. The Mitf-specific supershifted complex (arrow) is seen when specific monoclonal anti-Mitf antibody is added to the reactions. The specificity of DNA binding is confirmed by unlabeled wild-type probe competition and unlabeled mutant probe competition for each stimulation condition. Moreover, as shown in the far right panel, there is no difference in Mitf's ability to bind the mouse cathepsin K promoter in nuclear extract from day 8 wild-type and op/op osteoclasts (the latter derived by in vitro rescue with recombinant M-CSF).
(C) Anti-p300 immunoprecipitates from osteoclast nuclear extracts contained Mitf specifically after M-CSF stimulation (see arrow), but not when stimulation was carried out in the presence of MEK inhibitor or no stimulation. [C] indicates isotype matched control antibody. IgH represents the immunoglobulin heavy chain.
(D) p300 selectively associates with the upper MAPK-phosphorylated TFE3 band (see arrow) and not the lower TFE3 band from osteoclast lysates
Molecular Cell 2001 8, DOI: ( /S (01) )

6. Figure 5
M-CSF Unresponsiveness and Fusion Defect in Mitfmi/mi but Not MitfVGA9/VGA9 Osteoclasts
(A) Upper panels depict multinucleated wild-type osteoclasts that express high levels of TRAP. Mitf and TFE3 localize in the nucleus. The lower panels depict cultured Mitfmi/mi mutant osteoclasts that are primarily mononuclear and express low levels of TRAP. Mutant Mitf and TFE3 localize in the nucleus in these mononuclear osteoclasts.
(B) Mitf mutation blocks M-CSF response. During M-CSF-induced osteoclast differentiation (also in the presence of serum and RANKL), TRAP activity increases with time in wild-type, but not in Mitfmi/mi mutant, osteoclasts. The increase in TRAP activity is M-CSF dependent—despite the presence of RANKL—as shown for Day 7 cultures.
(C) Resorption assay on dentine bone slices. Wild-type mouse osteoclast cultures in the presence of RANKL and MCSF are capable of bone resorption, whereas the Mitfmi/mi mutant osteoclasts cannot resorb bone despite the presence of M-CSF (and RANKL).
(D) Calcium phosphate resorption assay: defects for dominant-negative, but not null Mitf mutant alleles. The Mitfmi allele encodes a protein which dominantly interferes with DNA binding by the family member TFE3 (Hemesath et al., 1994). The MitfVGA9 allele encodes a large, genetically null genomic deletion in the Mitf gene (Hodgkinson et al., 1993). Calcium phosphate resorption assays reveal a profound deficiency in Mitfmi/mi osteoclasts, whereas osteoclasts from the null mutant MitfVGA9/VGA9 display robust resorption. These data suggest a functional role for a related bHLH-ZIP family member (such as TFE3) in osteoclast function
Molecular Cell 2001 8, DOI: ( /S (01) )

7. Figure 6 Expression of S73A Mitf Mutant Decreases Osteoclast Fusion
(A) RAW preosteoclast cells were transfected with green fluorescent protein (GFP) and either wild-type Mitf, vector, or S73A Mitf and then were cultured in mouse osteoclast medium. After 3 days in culture, the number of multinucleated (greater than 2 nuclei/cell), transfected cells (GFP positive) were counted. The S73A Mitf mutant displayed impaired osteoclast fusion compared to wild-type Mitf.
(B) Representative pictures of transfected osteoclasts are shown
Molecular Cell 2001 8, DOI: ( /S (01) )