CD74, also known as invariant chain (Ii) is a type II transmembrane glycoprotein that is a component of the class II major histocompatibility complex (MHC). MHC class II molecules are synthesized and assembled in the endoplasmic reticulum (ER) through the non-covalent association of MHC α and β chains to trimers of invariant chain (Ii).1, 2 CD74 is expressed in class II positive cell types, including monocytes, B cells, activated T cells, and fibroblasts,3, 4 whereas macrophage migration inhibitory factor (MIF) is ubiquitously produced by a variety of cells including monocytes, endothelial cells, keratinocytes, anterior pituitary cells, and osteoblasts, suggesting their possible interaction within the immune system as well as multifunctional physiological effects.5–9 Ii is a nonpolymorphic glycoprotein that has diverse immunological functions.3 It associates with MHC class II in order to regulate trafficking in antigen-presenting cells (APCs) as well as to influence the differentiation of B lymphocytes.10, 11 CD74 serves as a cell surface receptor protein for MIF. When MIF binds to CD74, then CD44 is recruited to form a CD74-CD44 complex. This, in turn, induces the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI3Kase)/protein kinase B (AKT) signaling.1, 12–14 The MIF-CD74-CD44 complex is believed to regulate the signaling of leukocyte migration to sites of inflammation.
Four isoforms of the CD74 are produced through alternative splicing. The shortest isoform (p33, 33 kD) is the most predominant compared to the p35, p41, and p43 isoforms.15, 16 Two small isoforms (p33 and p35) are believed to be involved in regulating class II MHC antigen presentation, whereas the p41 isoform may play a key role in T cell selection in the thymus.15, 16
Osteoclasts (OCs) are multinucleated giant cells that originated from monocyte/macrophage lineage progenitor cells.17–20 They are the principal bone resorbing cells and have multiple characteristic features, such as multinucleation, expression of tartrate-resistant acid phosphatase (TRAP), calcitonin receptors (CTRs), vitronectin receptors (integrin αvβ3), and matrix metalloproteinase 9 (MMP 9).17, 21–23 The formation of multinucleated OCs is induced by fusion of mononuclear OC precursors. OC-mediated bone-resorbing activity is critical for bone remodeling.17, 21–24 The binding of receptor activator of NF-κB ligand (RANKL) to receptor activator of NF-κB (RANK) regulates OC differentiation and bone resorption.24–29 This binding initiates a signaling pathway for OC development and mediates the activation of mature OCs. RANKL-RANK binding induces various second messenger signals that mediate OC differentiation, such as nuclear factor of activated T cells cytoplasmic 1 (NFATc1), TRAP, and cathepsin K.30–33
We previously reported that MIF downregulated osteoclast-like cell (OCL) formation in bone marrow cultures and that bone marrow cells from MIF knockout (KO) mice formed a greater number of TRAP(+) OCL compared to those from WT mice in response to bone-resorbing cytokines.34 In the current study, we examined if CD74 is required for MIF to signal in osteoclastogenesis. We measured OC formation in vitro as well as the bone mass of WT and CD74-deficient mice. In addition, we examined the effect of MIF on the expression of c-fos and NFATc1 in bone marrow macrophage (BMM) cultures.
Subjects and Methods
All animals used in the experiments were 7-week-old to 9-week-old WT and CD74KO mice with in a C57BL/6J background. CD74KO mice were originally generated by replacing the first intron with a neomycin-resistant gene cassette to inactivate the CD74 gene.35 Heterozygous CD74KO mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA) and crossed to generate littermate WT and CD74KO mice. PCR genotyping assay was used to identify the mutant allele. Homozygous CD74KO mice appeared normal and are indistinguishable from WT littermates in their general health, growth rate, and breeding performances. Mice were housed in the Center for Comparative Medicine at the University of Connecticut Health Center. All animal protocols were approved by the Animal Care Committee of the University of Connecticut Health Center.
Bone marrow cell cultures
Mouse bone marrow cells were isolated from the femur and tibia by a modification of published methods.36–38 Briefly, bone marrow cells were flushed, collected, and washed twice with α modified essential medium (α-MEM). Cells were then cultured (5 × 104 cells/wells in 96-well plate) with complete α-MEM medium (10% heat-inactivated fetal bovine serum [HIFBS], 2 mM L-glutamine, 100 U/mL penicillin-streptomycin) in the presence of human macrophage colony-stimulating factor (hM-CSF) and/or human RANKL (hRANKL) (both at 30 ng/mL, gifts from Dr. Y. Choi, University of Pennsylvania) and with or without recombinant mouse MIF (rmMIF) (25 ng/mL; R&D Systems, Minneapolis, MN, USA). We also used bone marrow macrophage/monocyte cells (BMM). BMM cells were prepared by incubating total bone marrow cells overnight in complete α-MEM. Nonadherent cells were collected and mononuclear cells were prepared using Ficoll-Hypaque (GE Healthcare, Piscataway, NJ, USA) density gradient centrifugation. Interface between Ficoll-Hypaque and medium was collected and used for BMM culture.39–41
In vitro OC formation assay
Mouse bone marrow or BMM cells were cultured with M-CSF and RANKL (both at 30 ng/mL or dose indicated) and with or without rmMIF (25 ng/mL) for up to 6 days. In some experiments, we isolated OC precursor population from fresh bone marrow cells, as described,42 for OC formation assay in vitro. The medium was replenished every 3 days and cells were fixed with 2.5% glutaraldehyde in PBS for 15 minutes at room temperature prior to TRAP enzyme histochemistry using a commercial kit (Sigma, St. Louis, MO, USA). TRAP-positive cells that contained more than three nuclei were counted as OCLs.
Pit formation assay
To examine the ability of OCLs that formed from WT and CD74KO mice bone marrow cells to resorb bone, we performed pit formation assays by culturing cells on UV-sterilized devitalized cortical bone slices that were placed in the wells of a 96-well plate with M-CSF and RANKL (both at 30 ng/mL) for 14 days. The bone slices were fixed with 2.5% glutaraldehyde in PBS and stained for TRAP. Bone slices were sonicated in 0.25 M NH4OH to remove cells and then stained with 1% toluidine blue in 1% borax buffer to visualize resorption pits. Pit area per OCL was measured using the VIA-160 video image measurement system (Boeckeler Instruments, Tucson, AZ, USA).
Colony forming unit granulocyte-macrophage (CFU-GM) assay
Total bone marrow cells were plated on a 35-mm tissue culture dish in 1 mL of 1.5% methylcellulose supplemented with 20% HIFBS, 2% bovine serum albumin (BSA), and 1.0 ng/mL recombinant murine granulocyte-macrophage colony stimulating factor (GM-CSF) (R&D Systems) as the source of colony-stimulating activity. Cultures were maintained in a humidified chamber at 37°C for 7 days. The number of colonies (>40 cells) were counted at the end of incubation.43
The antibodies used for flow cytometric analysis are all commercially available. These include: anti-mouse CD45R (B220) for B-cell lineage cells; anti-mouse CD3 for T cell lineage cells; anti-mouse CD11b (Mac-1) for macrophage lineage cells; anti-mouse CD117 (c-kit), anti-mouse CD115 (c-fms), and anti-mouse CD74 (BD Biosciences, San Jose, CA, USA). Unless indicated, all antibodies and secondary step reagents were obtained directly conjugated to fluorochromes or biotinylated from commercial sources (eBiosciences, San Diego, CA, USA). Labeling of bone marrow cells for flow cytometric analysis was performed by standard staining procedures in 1× Hank's balanced salt solution (HBSS) (Gibco, Invitrogen Corp., Carlsbad, CA, USA) containing 0.01 M HEPES (pH 7.4) and supplemented with 2% fetal bovine serum (FBS). Bone marrow cells were collected by flushing the long bones with medium using a 25G needle. After washing, the red blood cells were lysed using ammonium chloride and the cell preparation was filtered through a nylon mesh and counted. The cells were kept on ice at all times. Dead cells were excluded by their ability to incorporate propidium iodide. Flow cytometric analysis was done on a FACSCalibur (BD Biosciences) and data analysis was performed using FlowJo software from Tree Star, Inc. (Ashland, OR, USA). Specific OC precursor population (B220− CD3− CD11b−/lo CD115+) was sorted in a BD-FACS Aria (BD Biosciences) equipped with five lasers and 18 fluorescence detectors.
Migration and cell proliferation assays
Cell migration assay was performed using modified Boyden chamber assay according to the manufacturer's recommendation (Cell Biolabs, Inc., San Diego, CA, USA). Briefly, either total bone marrow cells (1 × 106) or magnetic-activated cell sorting (MACS)-sorted triple negative fraction in the bone marrow (B220−CD3− Mac-1−, 2 × 105 cells) were placed in the upper chamber, which was separated from lower chamber by the polycarbonate membrane (8-µm pore size) in a 96-well plate. Cells were incubated in the presence or absence of HIFBS, the combination of M-CSF and RANKL and/or MIF in the medium with 10% HIFBS for 12 hours. Migratory cells passed through the membrane and attached to the bottom side of membrane. These cells were dissociated, lysed, and quantified using fluorescent dye.
Cell proliferation assay was performed using WST-1 reagent according to the manufacturer's recommendation (Roche, Mannheim, Germany). Briefly, bone marrow cells (5 × 104) were plated in a 96-well plate and treated with M-CSF and/or RANKL for up to 6 days prior to the assay.
Micro–computed tomography analysis
The femurs from WT and CD74KO mice were removed and fixed in 70% ethanol at 4°C for micro–computed tomography (microCT) analysis. Trabecular and cortical morphometry within the metaphyseal region of the distal femur was quantified using microCT (µCT40; Scanco Medical AG, Bassersdorf, Switzerland). Three-dimensional images were reconstructed using standard convolution back-projection algorithms with Shepp and Logan filtering, and rendered at a discrete density of 578, 704 voxels/mm3 (isometric 12-µm voxels). Threshold segmentation of bone from marrow and soft tissue was performed in conjunction with a constrained Gaussian filter to reduce noise. Volumetric regions for trabecular analysis were selected within the endosteal borders to include secondary spongiosa of the femur (1 mm from the growth plate and extending 1 mm proximally). Trabecular morphometry was characterized by measuring the bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular spacing (Tb.Sp). Cortical morphometry was analyzed within a 600-µm-long section at mid-diaphysis of the femur and included measurements of average thickness and cross-sectional area. The measurement terminology and units used for microCT analysis were those recommended by the Committee of the American Society for Bone and Mineral Research.44
Bone histomorphometry was performed on mouse long-bones (femurs) from WT and CD74KO male mice. Bone histomorphometric analysis was performed in a blinded, nonbiased manner using a computerized semiautomated system (Osteomeasure, Nashville, TN, USA) with light microscopy. The bones from at least 7 to 8 mice per group were examined. The quantification of OCs was performed in paraffin embedded tissues that were stained for TRAP. OCs were identified as multinucleated TRAP-positive cells adjacent to bone. The measurement terminology and units used for histomorphometric analysis were those recommended by the Nomenclature Committee of the American Society for Bone and Mineral Research.45 Briefly, all measurements were confined to the secondary spongiosa and restricted to an area between 400 and 2000 µm distal to the growth plate-metaphyseal junction of the distal femur.
RNA extraction and RT-PCR
Total RNA was extracted from either WT or CD74 KO BMM cells at the indicated time with TRI reagent (Molecular Research Center, Cincinnati, OH, USA) according to the manufacturer's recommendation.46 Total RNA was converted to cDNA by reverse transcriptase (High Capacity cDNA Reverse Transcription Kit; Applied Biosystems, Carlsbad, CA, USA) using random hexamers, and aliquots of RT mixtures were used for PCR amplification. PCR amplification was done using gene-specific PCR primers and Taq polymerase (AmpliTaq; Applied Biosystems). Specific primer sets were designed from published mRNA sequences: murine NFATc147 (forward: 5′-TGC AAC AAG CGC AAG TAC-3′; reverse: 5′-GTA GCG TGA GAG GTT CAT TCT-3′), murine c-fos48 (forward: 5′-TCT AGT GCC AAC TTT ATC CC-3′; reverse: 5′-AGT CAT CAA AGG GTT CTG C-3′), and murine GAPDH49 (forward: 5′-TGA AGG TCG GTG TGA ACG GAT TTG GC-3′; reverse: 5′-CAT GTA GGC CAT GAG GTC CAC CAC-3′). The amplified products were run in a 1.5% agarose gel, stained with ethidium bromide, and photographed under UV illumination.
Western blot analysis
BMM cells (5 × 105 cells/well in six multi well plate) were cultured with M-CSF and RANKL (both at 30 ng/mL) with or without MIF (25 ng/mL) for up to 6 days or treated with M-CSF alone for 3 days before the cells were treated with RANKL with or without rmMIF for the period indicated in each experiment. Cells were then washed with cold PBS twice before lysis buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 1% Triton X-100, 10% glycerol) containing protease and phosphatase inhibitors were added. Cell extracts were collected, applied to 8% to 10% SDS-PAGE gels and transferred onto nitrocellulose membranes by electroblotting. The membranes were blocked for 1 hour in a blocking buffer containing 5% powdered milk or 5% BSA in Tris-Buffered Saline and Tween 20 (TBS-T). The membranes were incubated with primary antibody overnight at 4°C followed by incubation with a secondary antibody conjugated to horseradish peroxidase (HRP). Reactive bands were detected by enhanced chemiluminescence using LumiGLO (Cell Signaling Technology, Danvers, MA, USA). Specific antibodies to c-fos and NFATc1 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) and antibodies to IκBα, phospho-c-jun, β-actin, and β-tubulin were purchased from Cell Signaling Technology.
Statistical analysis was performed by Student's t test or one-way analysis of variance (ANOVA) and the Bonferroni post hoc test when ANOVA demonstrated significant differences. All experiments were repeated at least twice and representative experiment or pooled data are shown.
CD74 protein expression in OC precursor population in bone marrow
In order to investigate if OCs can directly respond to MIF treatment, we examined the expression of CD74, which is a receptor for MIF, on bone marrow cells. CD74 expression was examined by flow cytometry using a fluorescein isothiocyanate (FITC)-conjugated antibody. We found that CD74 was expressed in approximately 18% of total bone marrow cells. It was enriched in the CD3−/B220− population as shown in Fig. 1A, indicating that the majority of lymphoid populations did not express this protein. Both CD11b (Mac-1)+ (40%) and TN (triple negative: B220−CD3− CD11b−) population (50%) contain CD74-expressing cells. We further analyzed CD74 expression in terms of CD115 (c-fms) and CD117 (c-kit) expression as reported42 (Fig. 1B.) In our previous report, we identified TN CD115+ CD117+ as the most efficient osteoclastogenic precursor population in the bone marrow. Interestingly, almost all CD115+ cells expressed CD74 in the TN population. This is significant because we previously identified TN CD115+ cells to contain highly efficient populations of OC precursor cells.42
MIF signals through CD74 in osteoclastogenesis in vitro
To investigate the role of MIF and its putative receptor, CD74, in osteoclastogenesis, we examined the ability of bone marrow cells from WT and CD74-deficient mice (CD74KO) to form OCs in vitro and the response of these cells to MIF treatment. CD74KO mice were generated by replacing the first intron of the CD74 gene with a neomycin cassette.35 Homozygous CD74KO mice appeared normal, and were indistinguishable from WT littermates in their general health, growth rate, and breeding performances. Bone marrow cells were cultured with M-CSF (30 ng/mL) and/or RANKL (30 ng/mL) and with or without MIF (25 ng/mL) for up to 6 days. As shown in Fig. 2A, bone marrow cells were cultured for 3 to 6 days with M-CSF and RANKL in vitro. OC formation peaked at day 5 and then decreased thereafter in cultures from both WT and CD74KO mice. However, the number of TRAP(+) OCLs formed in bone marrow cells from CD74KO mice was significantly greater than from WT mice at days 5 and 6. In a separate experiment we treated bone marrow cells from WT or CD74KO mice with various doses of RANKL for 5 days in the presence of M-CSF (Fig. 2B). The number of TRAP(+) OCLs that formed in bone marrow cells from CD74KO mice was greater than those from WT mice at 10 and 30 ng/mL of RANKL. Subsequently, we examined the effect of exogenous MIF on OCL formation in bone marrow cultures from WT and CD74KO mice. MIF inhibited OCL formation in 5-day bone marrow cultures from WT mice as described.34 However, there was no significant difference in the number of OCLs formed in cultures from CD74KO that were treated with exogenous MIF in the presence of M-CSF and RANKL (Fig. 2C). This result implies that the CD74 transmembrane protein is involved in the response of OC precursor cells to MIF. To determine if the effect of MIF is directly on OC precursor cells, we cultured BMMs from WT mice in the absence of stromal cells. As shown in Fig. 2D, the addition of exogenous MIF (25 ng/mL) to WT BMM cultures decreased OCL formation in vitro. Because the majority of CD74-expressing cells are in the OC precursor population, we FACS-sorted an OC precursor population (B220−CD3− CD11b−/lo CD115+), which, we have reported,42 contains the cells that are most efficient at differentiating into OCs. Our goal was to determine if cells from CD74KO mice had an altered potential to become OCLs. As shown in Fig. 2E, there was no significant difference in osteoclastogenic potential between OC precursor cell in bone marrow cells from WT and CD74KO mice when we plated cells at the same OC precursor cell density. This indicates that there was no significant difference between cells from WT and CD74KO mice in the potential to form OCLs in vitro. In addition, OCL formation was downregulated in bone marrow cells from WT mice in response to MIF treatment but not in CD74KO cells. Moreover, we examined the ability of MIF to affect the migration of cells in both total bone marrow and MACS-sorted TN (B220−CD3− CD11b−) population in WT mice (Supplemental Fig. 1) by modified Boyden chamber assay. MIF treatment did not affect the migration of cells in either the total (Supplemental Fig. 1A) or the TN fraction (Supplemental Fig. 1B) of cells from WT and CD74KO mice.
We also cultured bone marrow cells from WT and CD74KO mice on bone slices in the presence of M-CSF and RANKL (both at 30 ng/mL) and at the conclusion of culture (14 days) we measured pit area. As shown in Fig. 2F, pit area per OC in cultures of cells from CD74KO mice was greater than that of cells from WT mice. These results indicate that OCs formed from CD74KO cells had a greater capacity to form OCLs and to resorb bone in vitro.
Bone marrow cells from CD74KO mice contain more OC precursor cells
To investigate the cause of the increase in the number of OCLs in cultures from CD74KO mice, we measured the number of GM-CFUs in the bone marrow of WT and CD74KO mice (Fig. 3A). Cells derived from GM-CFUs, which uses granulocyte-macrophage colony-stimulating factor (GM-CSF) to expand populations, have the ability to form OCs with high frequency.43 As shown in Fig. 3A, bone marrow cells from CD74KO mice had a significant increase (26%) in GM-CFUs compared to those from WT. This result implies that the number of OC precursors in the bone marrow of CD74 KO mice was greater than in WT mice. Because we reported previously that most of the early osteoclastogenic activity of TN fraction was within cells with the phenotype (B220−CD3− CD11b−/lo) CD115 (c-fms)high CD117 (c-kit)high (population 4, P4),42 we examined if there was any significant difference in this particular fraction (P4) between WT and CD74KO mice. Interestingly, we found that there was a significant decrease in the P4 fraction percentage and absolute number in the bone marrow from CD74KO mice compared to WT mice (Fig. 3B). As with P4, there was also a significant decrease in the P5 [TN (B220−CD3− CD11b−/lo) CD115 (c-fms)high CD117 (c-kit)int] and P6 [TN (B220−CD3− CD11b−/lo) CD115 (c-fms)high CD117 (c-kit)−] fractions from CD74KO mice compared to WT mice (data not shown). We further analyzed the monocyte/macrophage population in the bone marrow (B220−CD3− CD11b+ CD115+) of WT and CD74KO mice. As shown in Fig. 3C, bone marrow cells from CD74KO mice contained a greater number of CD11b+ CD115+ cells (22%), which are also known to differentiate into OCs in vitro.42 Figure 3D depicts the total OC precursor population (CD11b−/lo CD115+ and CD11b+ CD115+ in the B220−CD3− gated population) in the bone marrow by FACS analysis and demonstrates that there are more OC precursors in CD74KO mice. It appears that the increase in the CD11b+ CD115+ population, which also can differentiate into OCs, accounts for the increase in GM-CFU cells that we observed in Fig. 3A. Subsequently, we examined if cells from CD74KO mice have altered proliferation potential using a WST-1 kit. WST-1 is a water-soluble tetrazolium salt and the rate of WST-1 cleavage by mitochondrial dehydrogenases correlates with the number of viable cells in the culture. There was no difference in the proliferation potential of bone marrow from WT and CD74KO mice (Supplemental Fig. 2) in response to M-CSF alone (Fig. 3A) or the combination of M-CSF and RANKL (Fig. 3B) for up to 6 days in vitro. These results (GM-CFU and flow cytometry) indicated that CD74KO mice contain a greater number of OC precursor cells in bone marrow compared to WT mice.
Bone phenotype of CD74KO mice are similar to MIF KO mice
We next performed microCT analysis to determine the bone mass of 8-week-old WT and CD74KO male and female mice. Both CD74KO mice and WT controls were in a C57BL/6J background. As shown in Fig. 4A, three-dimensional microstructural analysis using high-resolution microCT indicated that loss of CD74 expression resulted in a significant decrease in both trabecular and cortical bone volume. Representative images of microCT scans of WT and CD74KO femurs are shown in Fig. 4A. Male CD74KO mice had decreased trabecular bone volume (BV/TV, 26%) and trabecular thickness (Tb.Th, 24%), and there was no effect of CD74KO deletion on trabecular bone mass in females at 8 weeks old (Fig. 4B, C). However, we found significant decreases in cortical bone area and thickness in CD74KO mice (by 14% and 11%, respectively) compared to WT male mice (Fig. 4D, E). Additionally, both cortical bone area and cortical thickness in CD74KO female mice were significantly decreased compared to WT females.
To examine if the loss of bone mass in male CD74KO mice was due to an increased number of OCs, we performed static histomorphometric analysis. Figure 5A (upper panel) shows the representative microphotographs from WT and CD74KO mice. The lower panel of Fig. 5A shows TRAP(+) OCs adjacent to the trabeculae in the bone marrow cavity (arrows). CD74KO male mice had significantly lower BV/TV (by 39%) compared to WT mice (Fig. 5B), which confirmed the microCT analysis. This was associated with a decrease in Tb.Th and an increase in trabecular spacing in CD74KO mice (Fig. 5C, D). We also examined the osteoblast and OC parameters in the femur. There was no significant difference in osteoblast surface in femurs between WT and CD74KO mice (Fig. 5E). However, we found a significant increase in the OC surface (Fig. 5F) as well as the eroded surface (Fig. 5G) in CD74KO (by 80% and 96%, respectively) compared to WT. As shown in Fig. 3, there was an increase in OC precursor cells and the osteoclastogenic potential of bone marrow cells in CD74KO mice relative to WT. It appears that the increase in the OC precursor population in the bone marrow cavity led to an increase in the number of OCs in vivo. These data indicate that mice which lack the MIF receptor, CD74, are similar to MIFKO mice in their bone phenotype.
MIF downregulates osteoclastogenesis by modulating RANKL-induced NFATc1 expression
To determine how MIF modulates osteoclastogenesis, we examined the effect of MIF on secondary messengers that are critical for osteoclastogenesis, using RT-PCR and Western blot analysis. As shown in Fig. 6A, c-fos mRNA and protein expression peaked at day 2 and decreased thereafter in BMM cultures that were treated with M-CSF and RANKL. MIF treatment significantly reduced c-fos mRNA and protein levels at day 2 by 41% (Fig. 6A). In addition, NFATc1 mRNA and protein expression was prominent at days 3 and 4 of cultures that were treated with M-CSF and RANKL and decreased thereafter. MIF also downregulated NFATc1 expression at days 3 and 4 by 70% and 50%, respectively (Fig. 6A). In our subsequent experiments, we also analyzed mRNA and protein expression of c-fos and NFATc1 levels in BMM cultures from WT and CD74KO mice in response to MIF treatment. As expected, both c-fos and NFATc1 mRNA and protein levels in the BMM cultures from CD74KO mice were greater than from WT mice. MIF treatment downregulated both c-fos and NFATc1 mRNA level in BMM cultures from WT mice whereas MIF treatment did not affect both mRNA levels in BMM cultures from CD74KO mice (Fig. 6B, C). These results indicate that MIF alters RANK downstream signaling components, which are involved in OC differentiation, and in the absence of MIF-CD74 interaction RANK downstream events are enhanced, which increases OC differentiation. Hence, this implies a possible mechanism by which MIF downregulates RANKL-induced osteoclastogenesis in BMM cultures.
RANKL-RANK interaction initiates a variety of signaling pathways such as NF-κB, activator protein 1 (AP-1), and Ca2+, and these can individually or collectively regulate NFATc1 expression. We next investigated how MIF modulates the NFATc1 signaling pathway in RANKL-induced BMMs. BMMs were cultured with M-CSF alone for 3 days before RANKL and/or MIF were added to determine the effect of RANKL and/or MIF on the phosphorylation of IκBα and c-jun. As shown in Fig. 7A, IκBα degradation by RANKL treatment was detected in a time-dependent manner in BMM cultures and MIF treatment did not affect this degradation. We next examined the time course on c-jun activation. Phosphorylation of c-jun peaked at 15 minutes and remained elevated at 30 minutes after RANKL treatment (Fig. 7B). The combination of RANKL and MIF downregulated the phosphorylation of c-jun by 80% at 15 minutes compared to RANKL alone. These results suggest that MIF inhibits the expression of both NFATc1 and AP-1, which are critical signals for OC differentiation, through effects that involve decreased activation of c-jun.
In this work we demonstrated that MIF requires the CD74 receptor to mediate its effects in osteoclastogenesis in bone marrow cultures. In addition, the bone phenotype of CD74KO mice phenocopied to a large degree that of MIFKO mice. Confirming the mirroring of responses of MIFKO and CD74KO cells, we found that bone marrow and BMM cultures from CD74KO mice had an increased capacity to form OCs and an increased number of OC precursor cells as measured by CFU-GM and flow cytometry. Consistent with these results, we also found that trabecular bone volume measured by microCT and static histomorphometric analyses was decreased in femurs from CD74KO mice compared to those from WT mice. In addition, the absence of CD74 was associated with an increase in the number of TRAP(+) OCs and eroded area on the surface of trabecular bone from femurs as measured by histomorphometry. In addition, CD74 expression was detected in the majority of OC precursor populations (TN CD115+) in the bone marrow. Based on these data, we speculate that MIF may directly inhibit osteoclastogenesis through interactions with CD74 on OC precursor cells.
In the current study, we attempted to elucidate if the downregulation of osteoclastogenesis in vitro was mediated through CD74. Our results showed that bone marrow cultures from CD74KO mice did not respond to MIF treatment, confirming that MIF requires this putative receptor to affect cell function. In addition, we found that OC precursor cells contain the majority of the CD74 expression on bone marrow cells, implying that MIF directly affects osteoclastogenesis through cell autonomous effects. Our data also imply that a deficiency in the MIF receptor, CD74, caused the increase in OC formation in vivo that we measured by static histomorphometric analysis. In summary, these findings indicate that CD74KO mice have an osteopenic bone phenotype that is similar to that of MIFKO mice. However, the bone phenotype of CD74KO mice resulted from the increased OC activity whereas that of MIFKO mice resulted from inhibited function of both OCs and osteoblasts.34 Clearly, there are major differences in how MIFKO and CD74KO mice arrive at low bone mass. This argues that there may be additional functions of MIF and CD74, possibly involving other cell types, that are independent of their direct effects on OCs. As shown in in vitro and in vivo studies of CD74KO mice, the number of OCs formed in vitro in bone marrow or BMM cell cultures was greater in the CD74KO mice compared to age- and gender-matched WT mice. The increase in the number of OCLs from the cultures from CD74KO mice was due to the increase in OC precursor cells in the bone marrow as measured by CFU-GM and flow cytometry. We believe that the increase in OC precursor was not due to a significant change in lymphocytes but macrophages in the bone marrow. In the bone marrow, there was a trend toward an increase in CD11b+ population in the bone marrow of CD74KO mice (34.9 ± 1.51%) compared to WT mice (31.6 ± 1.55%, p = 0.07, data not shown). However, there could be additional ligands for the CD74 receptor, and CD74 could influence the function of other cell types that, in turn, regulate OC precursor abundance. We also found that there was no difference in the potential to become OCs in the bone marrow cells from WT and CD74KO mice when highly purified FACS-sorted OC precursor cells were cultured at an equal density (Fig. 2E).
In contrast to its initially reported function of MIF, which prevents the random migration of macrophages,46 we failed to demonstrate that MIF modulates the migration or replication of cells in our culture system, as shown in Supplemental Figs. 1 and 2. Additionally, time-lapse imaging of OC differentiation, using BMM cultures, indicated that MIF treatment did not affect the mobility of cells in BMM cultures (data not shown).
As in MIFKO mice, only male CD74KO mice showed a distinct osteopenic bone phenotype at 8 weeks of age. However, the cortical parameters, cortical bone area and thickness, were lower in both male and female CD74KO mice compared to gender-matched WT mice. We also examined the OC surface (Oc.S/BS) of CD74KO male mice in vivo and found that there was a significant increase in the OC surface and eroded surface compared to WT mice.
In this study we also determined whether MIF affects RANKL-induced signaling mechanisms that are critical for osteoclastogenesis. Binding of RANKL to its signaling receptor RANK also results in the activation of c-jun NH2 terminal kinase (JNK), a mitogen-activated protein (MAP) kinase, which increases the transactivating activity and production of AP-1 (c-fos and c-jun) transcription factors. Studies of RANK signaling in model cell lines have demonstrated that the binding of multiple tumor necrosis factor (TNF)-associated factors (TRAF) proteins to distinct regions in the RANK cytoplasmic domain50, 51 is a critical step in JNK activation.52 NFATc1 is the critical transcription factor for RANKL-induced osteoclastogenesis. Expression of NFATc1 is dependent on both the TRAF6 and the c-fos pathway. NFATc1 and c-fos synergistically induce the expression of OC-specific genes.47, 53–57 Moreover, NFATc1 has been known to be critical for RANKL-induced osteoclastogenesis through three separate pathways: AP-1, NF-κB, and calcium signaling.58
To date, MIF has been reported to inhibit apoptosis and promote tumor cell survival through the AKT pathway59 in various cell types. In addition, MIF has been implicated to mediate the regulation of CD74-dependent ERK MAP kinase (MAPK) signaling and the activation of cytosolic phospholipase A2.1, 60, 61 It also modulates the activities of the tumor-associated protein c-jun activation domain-binding protein-1 (JAB1) and signal through the COP9 signalosome (CSN).62, 63 This study is the first to report that MIF modulates RANKL-induced osteoclastogenic signaling mechanisms. As shown in Fig. 6, RANKL-induced c-fos and NFATc1 mRNA and protein expression in BMM cultures were significantly downregulated by MIF treatment. It is also known that the expression of c-fos is followed by NFATc1 expression. Interestingly, MIF does not appear to regulate NFATc1 expression through the NF-κB pathway because we found no effect of MIF treatment on IκBα. Hence, these data imply that the downregulation of OC formation in vitro by MIF resulted from suppression of AP-1 expression and subsequently, NFATc1 expression.
In conclusion, our findings imply that MIF signals through the CD74 receptor and that the bone phenotype of CD74KO mice is similar to MIFKO mice. MIF also downregulated OC formation by suppressing the activity of AP-1, followed by suppressing NFATc1 activation. We also demonstrated for the first time that MIF requires CD74 on the surface of OC precursors to suppress RANKL-induced osteoclastogenesis. We conclude that CD74 is essential for MIF to affect RANK downstream signaling and regulate bone mass.
All authors state that they have no conflicts of interest.
This study was supported by the U.S. Department of Health and Human Services, National Institutes of Health, National Institutes of Arthritis and Musculoskeletal Diseases (R01-AR055143 to SKL). We thank Dr. Joseph A Lorenzo (University of Connecticut Health Center) for his critical reading of the manuscript.
Authors' roles: SHM performed the majority of experiments; HYW, PH, and HLA performed flow cytometry and FACS sorting; SHM, HLA, and SKL designed the project, discussed the work, and wrote the manuscript.