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. Author manuscript; available in PMC: 2014 Apr 1.
Published in final edited form as: J Bone Miner Res. 2013 Apr;28(4):948–959. doi: 10.1002/jbmr.1787

Deletion of CD74, a putative MIF receptor, in mice enhances osteoclastogenesis and decreases bone mass

Se Hwan Mun 1, Hee Yeon Won 1, Paula Hernandez 1, Hector Leonardo Aguila 2, Sun-Kyeong Lee 1
PMCID: PMC3563845  NIHMSID: NIHMS413830  PMID: 23044992

Abstract

CD74 is a type II transmembrane protein that can act as a receptor for macrophage migration inhibitory factor (MIF) and plays a role in MIF-regulated responses. We reported that MIF inhibited osteoclast formation and MIF KO mice had decreased bone mass. We therefore examined if CD74 was involved in the ability of MIF to alter osteoclastogenesis in cultured bone marrow (BM) from WT and CD74 deficient (KO) male mice. We also measured the bone phenotype of CD74 KO male mice. Bone mass in the femur of 8 week old mice was measured by micro-computed tomography and histomorphometry.

Bone marrow cells from CD74 KO mice formed 15% more osteoclast like cells (OCL) with M-CSF and RANKL (both at 30 ng/ml) compared to WT. Addition of MIF to WT cultures inhibited OCL formation by 16% but had no effect on CD74KO cultures. The number of colony forming unit granulocyte-macrophage (CFU-GM) in the bone marrow of CD74 KO mice was 26% greater than in WT controls. Trabecular bone volume (TBV) in the femurs of CD74 KO male mice was decreased by 26% compared to WT. In addition, cortical area and thickness were decreased by 14% and 11%, respectively. Histomorphometric analysis demonstrated that TRAP(+) osteoclast number and area were significantly increased in CD74 KO by 35% and 43%, respectively compared to WT. Finally, we examined the effect of MIF on RANKL-induced-signaling pathways in BMM cultures. MIF treatment decreased RANKL-induced NFATc1 and c-Fos protein in BMM cultures by 70% and 41%, respectively.

Our data demonstrate that CD74 is required for MIF to affect in vitro osteoclastogenesis. Further, the bone phenotype of CD74 KO mice is similar to that of MIF KO mice. MIF treatment of WT cultures suppressed RANKL-induced AP-1 expression, which resulted in decreased osteoclast differentiation in vitro. We propose that CD74 plays a critical role in the MIF inhibition of osteoclastogenesis.

INTRODUCTION

CD74, also known as invariant chain (Ii) is a type II transmembrane glycoprotein which 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) while 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 non-polymorphic glycoprotein, which has diverse immunological functions (3). It associates with MHC class II in order to regulate trafficking in antigen presenting cells (APC) 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 ERK1/2 and PI3Kase/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 (33kD, p33) 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 while the p41 isoform may play a key role in T cell selection in the thymus (15, 16).

Osteoclasts (OCs) are multinucleated giant cells, which originated from monocyte/macrophage lineage progenitor cells (17-20). They are the principal bone resorbing cell and have multiple characteristic features such as multinucleation, expression of tartrate-resistant acid phosphatase (TRAP), calcitonin receptors (CTR), vitronectin receptors (integrin αvβ3) as well as matrix metalloproteinase (MMP) 9 (17, 21-23). The formation of multinucleated OCs is induced by fusion of mononuclear osteoclast precursors. OC-mediated bone resorbing activity is critical for bone remodeling (17, 21-24). The binding of RANKL to RANK regulates OC differentiation and bone resorption (24-29). This binding initiates a signaling pathway for osteoclast development and mediates the activation of mature OCs. RANKL-RANK binding induces various second messenger signals that mediate osteoclast differentiation such as the nuclear factor of activated T cells c1 (NFATc1), tartrate-resistant acid phosphatase (TRAP) and cathepsin K (30-33).

We previously reported that MIF down-regulated OCL formation in bone marrow cultures and that bone marrow cells from MIF 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 osteoclast 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.

MATERIAL AND METHODS

Animals

All mice used in the experiments were seven to nine (7 - 9) weeks old WT and CD74KO and in a C57BL/6J background. CD74KO mice was originally generated by replacing the first intron with neomycin resistant gene cassette to inactivate the CD74 gene (35). Heterozygous CD74 KO mice were purchased from Jackson Laboratories (Bar Harbor, ME) 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 as well as their 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 previously published methods (36-38). Briefly, bone marrow cells were flushed, collected and washed twice with 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 hM-CSF and/or hRANKL (both at 30 ng/ml, gifts from Dr. Y. Choi, University of Pennsylvania) and with or without rmMIF (25 ng/ml, R and D Systems, Minneapolis, MN). We also used bone marrow macrophage/monocyte cells (BMM). BMM cells were prepared by incubating total bone marrow cells overnight in complete α-MEM. Non-adherent cells were collected and mononuclear cells were prepared using Ficoll-Hypaque (GE Healthcare, Piscataway, NJ) density gradient centrifugation. Interface between Ficoll-Hypaque and medium was collected and used for BMM culture (39-41).

In vitro osteoclast 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 osteoclast precursor population from fresh bone marrow cells as described (42) for osteoclast formation assay in vitro. The medium was replenished every 3 days and cells were fixed with 2.5% glutaraldehyde in PBS for 15 min at room temperature prior to TRAP enzyme histochemistry using a commercial kit (SIGMA, St. Louis, MO). TRAP-positive cells that contained more than 3 nuclei were counted as osteoclast-like cells.

Pit formation assay

To examine the ability of OCL 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 0.25M 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).

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% BSA and 1.0 ng/ml recombinant murine GM-CSF (R & D Systems, Minneapolis, MN) as the source of colony-stimulating activity. Cultures were maintained in a humidified chamber at 37 °C for 7 days. The numbers of colonies (more than 40 cells) were counted at the end of incubation (43).

Flow cytometry

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) and anti-mouse CD115 (c-fms) and anti-mouse CD74 (BD Biosciences, San Jose, CA). Unless indicated, all antibodies and secondary step reagents were obtained directly conjugated to fluorochromes or biotinylated from commercial sources (eBiosciences, San Diego, CA). Labeling of bone marrow cells for flow cytometric analysis was performed by standard staining procedures in 1x HBSS (Hank’s balanced salt solution, GIBCO, Invitrogen Corp., Carlsbad, CA) 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 25-gauge needle. After washing, the red blood cells were lysed using ammonium chloride and the cell preparation 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 and data analysis was performed using FlowJo software from Tree Star, Inc. (Ashland, OR). Specific osteoclast precursor population (B220− CD3− CD11b−/lo CD115+) was sorted in a BD-FACS Aria (BD Biosciences. San Jose, CA, USA) 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 manufacturer’s recommendation (Cell Biolabs, Inc, San Diego, CA). Briefly, either total bone marrow cells (1 × 106 cells) or 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 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.

MicroCT analysis

The femurs from WT and CD74KO mice were removed and fixed in 70% ethanol at 4°C for micro-CT analysis. Trabecular and cortical morphometry within the metaphyseal region of the distal femur was quantified using micro-CT (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 measurements terminology and units used for microCT analysis, were those recommended by the Committee of the American Society for Bone and Mineral Research (44).

Histomorphometric analysis

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) with light microscopy. The bones from at least 7-8 mice per group were examined. The quantification of osteoclasts was performed in paraffin embedded tissues that were stained for TRAP. Osteoclasts were identified as multinucleated TRAP-positive cells adjacent to bone. The measurements 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) according to manufacturer’s recommendation (46). Total RNA was converted to cDNA by reverse transcriptase (High Capacity cDNA Reverse Transcription Kit, Applied Biosystems) using random hexamer 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, Carlsbad, CA). Specific primer sets were designed from published mRNA sequences: murine NFATc1 (47) (forward: 5′-TGC AAC AAG CGC AAG TAC-3′; reverse: 5′-GTA GCG TGA GAG GTT CAT TCT-3′), murine c-fos (48) (forward: 5′-TCT AGT GCC AAC TTT ATC CC-3′; reverse: 5′-AGT CAT CAA AGG GTT CTG C-3′), murine GAPDH (49) (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 6 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 8-10% 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 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). Specific antibodies to c-fos and NFATc1 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and antibodies to IκBα, phospho-c-jun, β-actin and β-tubulin were purchased from Cell Signaling Technology.

Statistical analysis

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.

RESULTS

CD74 protein expression in osteoclast precursor population in bone marrow

In order to investigate if osteoclasts 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 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 figure 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 previously reported (42) (Figure 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 since we previously identified TN CD115+ cells to contain highly efficient populations of osteoclast precursor cells (42).

Figure 1.

Figure 1

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CD74 expression in bone marrow cells. Bone marrow cells were analyzed using multiple antibodies for surface antigen (CD3, B220, CD11b, CD115, CD117 and CD74). (A) CD74 expression was analyzed in lymphoid cells, CD3− B220− CD11b−/lo (triple negative, TN), and CD11b+ fractions. (B) CD74 expression in TN population, which are divided into two fractions with CD115 (c-fms) and CD117 (c-kit). The majority of CD115+ fraction expressed CD74 and approximately 40% of CD115-population expressed CD74 in TN fraction.

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 wild type (WT) and CD74 deficient mice (CD74KO) to form osteoclasts in vitro and the response of these cells to MIF treatment. CD74KO mice were generated by replacing the 1st intron of 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 figure 2A bone marrow cells were cultured for 3-6 days with M-CSF and RANKL in vitro. Osteoclast formation peaked at day 5 and then decreased thereafter in cultures from both WT and CD74KO mice. However, the number of TRAP(+) OCL cells formed in bone marrow cells from CD74KO mice was significantly greater than from WT mice at day 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 (Figure 2B). The number of TRAP(+) OCL 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 previously (34). However, there was no significant difference in the number of OCL formed in cultures from CD74KO that were treated with exogenous MIF in the presence of M-CSF and RANKL (Figure 2C). This result implies that the CD74 transmembrane protein is involved in the response of osteoclast precursor cells to MIF. To determine if the effect of MIF is directly on osteoclast precursor cells, we cultured bone marrow macrophage (BMM) from WT mice in the absence of stromal cells. As shown in figure 2D, the addition of exogenous MIF (25 ng/ml) to WT BMM cultures decreased OCL formation in vitro. Since the majority of CD74 expressing cells are in the osteoclast precursor population, we FACS-sorted an osteoclast precursor population (B220− CD3− CD11b−/lo CD115+), which, we previously reported (42), contains the cells that are most efficient at differentiating into osteoclasts. Our goal was to determine if cells from CD74KO mice had an altered potential to become OCL. As shown in figure 2E, there was no significant difference in osteoclastogenic potential between osteoclast precursor cell in bone marrow cells from WT and CD74KO mice when we plated cells at a same osteoclast precursor cell density. This indicates that there was no significant difference between cells from WT and CD74KO mice in the potential to form OCL in vitro. In addition, OCL formation was down-regulated 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 triple negative (B220− CD3− CD11b−) population in WT mice (Supplemental figure 1) by modified Boyden chamber assay. MIF treatment did not affect the migration of cells in either the total (Supplemental figure 1A) or the triple negative fraction (Supplemental figure 1B) of cells from WT and CD74KO mice.

Figure 2.

Figure 2

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TRAP (+) OCL formation assay. (A) Bone marrow cells from WT and CD74KO mice were cultured with M-CSF and RANKL (both 30 ng/ml) up to 6 days and TRAP stained. (B) Bone marrow cells from WT and CD74KO were treated with RANKL at a various doses (3, 10, 30 ng/ml) for 5 days. (C) Bone marrow cells from WT and CD74KO were cultured with M-CSF and RANKL (both at 30 ng/ml) and/or MIF (25 ng/ml) for 5 days and TRAP stained. (D) Bone marrow macrophage cells from WT mice were cultured with M-CSF, RANKL (both at 30 ng/ml) and/or MIF (25 ng/ml) for up to 6 days and TRAP stained. (E) FACS-sorted triple negative (TN) CD115 (c-fms)+ bone marrow cells from WT and CD74KO mice were cultured with M-CSF, RANKL (both 30 ng/ml) and/or MIF (25 ng/ml) for up to 6 days and TRAP stained. (F) Bone marrow cells from WT and CD74KO mice were cultured with M-CSF and RANKL (both at 30 ng/ml) on bone slices for 14 days. Bone resorption area formed by OCL from CD74KO mice was greater than that from WT mice. Values represent mean±SEM. NS: Not significant. *, Significant effect of CD74KO mice, p<0.05. #, Significant effect of MIF treatment, p<0.05.

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 figure 2F, pit area per osteoclast in cultures of cells from CD74KO mice was greater than that of cells from WT mice. These results indicate that osteoclasts formed from CD74KO cells had a greater capacity to form OCL and to resorb bone in vitro.

Bone marrow cells from CD74KO mice contain more osteoclast precursor cells

To investigate the cause of the increase in OCL numbers in cultures from CD74KO mice, we measured the number of colony-forming unit granulocyte-macrophage (CFU-GM) in the bone marrow of WT and CD74KO mice (Figure 3A). Cells derived from CFU-GM, which uses GM-CSF to expand populations, have the ability to form osteoclasts with high frequency (43). As shown in figure 3A, bone marrow cells from CD74KO mice had a significant increase (26%) in CFU-GM compared to those from WT. This result implies that the number of osteoclast precursor in the bone marrow of CD74 KO mice was greater than in WT mice. Since 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 (Figure 3B). As with P4, there was also a significant decreases 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 CD74 KO mice compared to WT mice (data not shown.) We further analyzed monocyte/macrophage population in the bone marrow (B220− CD3− CD11b+ CD115+) of WT and CD74KO mice. As shown in figure 3C, bone marrow cells from CD74KO mice contained a greater number of CD11b+ CD115+ cells (22%), which are also known to differentiate into osteoclast in vitro (42). Figure 3D depicts the total osteoclast 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 osteoclast precursors in CD74KO mice. It appears that the increase in the CD11b+ CD115+ population, which also can differentiate into osteoclast, accounts for the increase in CFU-GM cells that we observed in Figure 3A. Subsequently, we examined if cells from CD74KO mice have altered proliferation potential using 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 figure 2) in response to M-CSF alone (A) or the combination of M-CSF and RANKL (B) for up to 6 days in vitro. These results (CFU-GM and FACS analysis) indicated that CD74KO mice contain a greater number of osteoclast precursor cells in bone marrow compared to WT mice.

Figure 3.

Figure 3

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(A) CFU-GM analysis. Bone marrow cells from WT and CD74KO mice (both 8 weeks old) were cultured in semisolid methylcellulose to examine the number of osteoclast precursor cells in the presence of GM-CSF (1 ng/ml) for 7 days and the number of colony formation was scored. (B) Distribution of P4 (B220− CD3− CD11b−/lo CD115hi CD117hi) population in total bone marrow of WT and CD74KO mice. (C) Distribution of CD11b+ CD115hi population in total bone marrow of WT and CD74 KO mice. Populations were gated for B220− CD3−. (D) Total osteoclast precursor population in bone marrow from WT and CD74KO mice. Bone marrow cells were B220− CD3− gated before the analysis. Bone marrow cells from CD74KO mice contained greater number of osteoclast precursor cells compared to WT mice. Values represent mean±SEM. *, Significant effect of CD74KO mice, p<0.05.

Bone phenotype of CD74KO mice are similar to MIF KO mice

We next performed micro-computed tomography (micro-CT) analysis to determine the bone mass of 8 week old wild type and CD74 KO male and female mice. Both CD74 KO mice and WT controls were in a C57BL/6J background. As shown in figure 4A, three-dimensional microstructural analysis using high-resolution microcomputed tomography indicated that loss of CD74 expression resulted in a significant decrease in both trabecular and cortical bone volume. Representative images of micro-CT scan of WT and CD74KO femurs are shown in figure 4A. Male CD74KO mice had decreased trabecular bone volume (BV/TV, 26%) and trabecular thickness (Tb. Th, 24%) while there was no effect of CD74KO deletion on trabecular bone mass in females at 8 week old (Figures 4B and 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 (Figures 4 D and E). Additionally, both cortical bone area and cortical thickness in CD74 KO female mice were significantly decreased compared to WT females.

Figure 4.

Figure 4

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Micro-CT analysis. The isolated femurs from WT and CD74KO mice (both 8 weeks old, n=7-8/group) were analyzed by Micro-CT. (A) Representative images of trabecular and cortical bone of WT and CD74KO male mice. (B) The trabecular bone volume (BV/TV) (C) The trabecular thickness (Tb. Th), (D) The cortical bone area and (E) the cortical thickness (Cortical Th). Values represent mean±SEM. *, Significant effect of CD74KO mice, p<0.05.

To examine if the loss of bone mass in male CD74 KO mice was due to an increased number of osteoclasts, we performed static histomorphometric analysis. Figure 5A is the representative microphotographs from WT and CD74KO mice (upper panel). Lower panel shows TRAP(+) osteoclast adjacent to the trabeculae in the bone marrow cavity (arrows). CD74 KO male mice had significantly less trabecular bone volume (by 39%) compared to WT mice (Figure 5B), which confirmed the micro-CT analysis. This was associated with a decrease in trabecular thickness and an increased in trabecular spacing in CD74 KO mice (Figures 5C and D). We also examined the osteoblast and osteoclast parameters in the femur. There was no significant difference in osteoblast surface in femurs between WT and CD74KO mice (Figure 5E). However, we found a significant increase in osteoclast surface (Figure 5F) as well as eroded surface (Figure 5G) in CD74 KO (by 80% and 96%, respectively) compared to WT. As shown in figure 3, there was increase in osteoclast precursor cells and the osteoclastogenic potential of bone marrow cells in CD74KO mice relative to WT. It appears that the increase in osteoclast precursor population in the bone marrow cavity led to an increase in osteoclast number in vivo. These data indicate that mice, which lack the MIF receptor, CD74, are similar to MIFKO mice in their bone phenotype.

Figure 5.

Figure 5

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Histomorphometric analysis. (A) Representative images of femurs of male WT and CD74KO mice that were stained with TRAP. TRAP(+) osteoclasts are marked with arrows in lower panel. (B) trabecular bone volume per tissue volume (BV/TV); (C) trabecular thickness (Tb.Th); (D) trabecular spacing (Tb. Sp); (E) osteoblast surface (Ob.S/BS); (F) osteoclast surface (Oc.S/BS) and (G) eroded surface (ES/BS). Values represent mean±SEM. *, Significant effect of CD74KO mice, p<0.05.

MIF down-regulates 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 figures 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% (Figures 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 down-regulated NFATc1 expression at day 3 and 4 by 70% and 50%, respectively (Figure 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 down regulated both c-fos and NFATc1 mRNA level in BMM cultures from WT mice while MIF treatment did not affect both mRNA levels in BMM cultures from CD74 KO mice (Figures 6B and C). These results indicate that MIF alters RANK downstream signaling components, which are involved in osteoclast differentiation and in the absence of MIF-CD74 interaction, RANK downstream events are enhanced, which increases osteoclast differentiation. Hence, this implicates a possible mechanism by which MIF down-regulates RANKL-induced osteoclastogenesis in BMM cultures.

Figure 6.

Figure 6

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Expression of c-fos and NFATc1 by RT-PCR and western blot analysis in BMM cultures. (A) BMM cells from WT were cultured and examined for c-fos and NFATc1 mRNA and protein expression in response to MIF treatment. BMM cells were cultured M-CSF and RANKL (both at 30 ng/ml) and/or MIF (25 ng/ml) for up to 6 days. Total RNA was extracted, reverse transcribed and the equal amount of cDNA mixture was subjected to PCR amplification. The equal amount of whole cell lysates of BMM was subjected to western blot analysis using c-fos and NFATc1 specific antibodies. β-Tubulin or actin was used as internal control. Bar graphs (lower panel) indicate the densitometric analysis of multiple experiments measuring c-fos and NFATc1 protein levels. c-fos (B) and NFATc1 (C) mRNA and protein levels in BMM cultures from WT and CD74KO mice. CD74 antibody was used to determine the absence of CD74 protein expression in CD74KO mice. Values represent mean±SEM. #, Significant effect of MIF treatment, p<0.05.

RANKL-RANK interaction initiates a variety of signaling pathways such as NF-κB, 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 BMM cells. 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 IkBα and c-jun. As shown in figure 7A, IkBα 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 (Figure 7B). The combination of RANKL and MIF down-regulated the phosphorylation of c-jun by 80% at 15 minutes compared to RANKL alone. These results suggest that MIF inhibit the expression of both NFATc1 and AP-1, which are critical signals for osteoclast differentiation, through effects that involve decreased activation of c-jun.

Figure 7.

Figure 7

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Activation of RANKL-induced IkBα and AP-1 were detected by western blot analysis. BMM from WT were cultured with M-CSF (30 ng/ml) for 3 days and were treated RANKL (30 ng/ml) and/or MIF (25 ng/ml) for up to 30 minutes before cells were lysed. The equal amount of whole cell lysates was subjected to western blot analysis using specific IkBα and phospho-c-jun antibodies. Representative western blot of degradation of IkBα (A) and phosphorylation of c-jun (B) was shown. Values represent mean±SEM. #, Significant effect of MIF treated, p<0.05.

DISCUSSION

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 MIF KO mice. Confirming the mirroring of responses of MIF KO and CD74 KO cells, we found that bone marrow and bone marrow macrophage cultures from CD74 KO mice had an increased capacity to form osteoclasts and an increased osteoclast precursor cell numbers as measured by CFU-GM and flow cytometry. Consistent with these results, we also found that trabecular bone volume measured by micro-CT and static histomorphometric analyses was decreased in femurs from CD74 KO mice compared to those from WT mice. In addition the absence CD74, was associated with an increase in the number of TRAP (+) osteoclasts 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 osteoclast 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 osteoclast precursor cells.

In the current study, we attempted to elucidate if the down-regulation 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 osteoclast 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 osteoclast 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 MIF KO mice. However, the bone phenotype of CD74KO mice resulted from the increased osteoclast activity while that of MIF KO mice resulted from inhibited function of both osteoclast and osteoblast (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 osteoclasts. As shown in in vitro and in vivo studies of CD74KO mice, the number of osteoclasts formed in vitro in bone marrow or bone marrow macrophage cell cultures was greater in the CD74KO mice compared to age and gender matched WT mice. The increase in OCL number from the cultures from CD74KO mice was due to the increase in osteoclast precursor cells in the bone marrow as measured by CFU-GM and flow cytometry. We believe that the increase in osteoclast 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 osteoclast precursor abundance. We also found that there was no difference in the potential to become osteoclasts in the bone marrow cells from WT and CD74KO mice when highly purified FACS-sorted osteoclast precursor cells were cultured at an equal density(Figure 2E).

In contrast to its initially reported function of MIF, which prevents the random migration of macrophages (46), we failed to demonstrate MIF to modulate the migration or replication of cells in our culture system as shown in supplemental figures 1 and 2. Additionally, time-lapse imaging of osteoclast differentiation, using BMM cultures, indicated that MIF treatment did not affect the mobility of cells in BMM cultures (data not shown).

As in MIF KO mice, only male CD74 KO 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 osteoclast surface (Oc.S/BS) of CD74KO male mice in vivo and found that there was a significant increase in the osteoclast surface and eroded surface compared to WT mice.

In this study we also determined if 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 MAP kinase, which increases the trans-activating 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 TNF-associated factors (TRAF) proteins to distinct regions in the RANK cytoplasmic domain (50, 51) is a critical step in JNK activation (52). Nuclear factor of activated T cells (NFAT) c1 (NFAT2) 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 osteoclast specific genes (47, 53-57). Moreover, NFATc1 has been known to be critical for RANKL-induced osteoclastogenesis through three separate pathways AP-1, NF-kB and calcium signaling (58).

To date MIF has been reported to inhibit apoptosis and promote tumor cell survival through the AKT pathway (59) in various cell types. In addition, MIF has been implicated to mediate the regulation of CD74-dependent ERK mitogen-activated protein 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 figure 6, RANKL induced c-fos and NFATc1 mRNA and protein expression in bone marrow macrophage cultures were significantly down-regulated 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-kB pathway since we found not effect of MIF treatment on IkBα. Hence, these data imply that the down-regulation of osteoclast 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 the bone phenotype of CD74KO mice is similar to MIFKO mice. MIF also down-regulated osteoclast 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 osteoclast precursors to suppress RANKL induced osteoclastogenesis. We conclude that CD74 is essential for MIF to affect RANK downstream signaling and regulate bone mass.

Supplementary Material

Supp FigureS1-S2

Acknowledgments

This study was supported by United States Public Health Services, National Institutes of Health, National Institutes of Arthritis and Musculoskeletal Diseases grants R01-AR055143. We thank Dr. Joseph A. Lorenzo (University of Connecticut Health Center) for his critical reading of the manuscript.

Funding Sources: Supported by United States Public Health Service, National Institutes of Health, National Institutes of Arthritis and Musculoskeletal Diseases. R01-AR055143 (SKL)

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