Augmenting MNK1/2 activation by c-FMS proteolysis promotes osteoclastogenesis and arthritic bone erosion

Osteoclasts are bone-resorbing cells that play an essential role in homeostatic bone remodeling and pathological bone erosion. Macrophage colony stimulating factor (M-CSF) is abundant in rheumatoid arthritis (RA). However, the role of M-CSF in arthritic bone erosion is not completely understood. Here, we show that M-CSF can promote osteoclastogenesis by triggering the proteolysis of c-FMS, a receptor for M-CSF, leading to the generation of FMS intracellular domain (FICD) fragments. Increased levels of FICD fragments positively regulated osteoclastogenesis but had no effect on inflammatory responses. Moreover, myeloid cell-specific FICD expression in mice resulted in significantly increased osteoclast-mediated bone resorption in an inflammatory arthritis model. The FICD formed a complex with DAP5, and the FICD/DAP5 axis promoted osteoclast differentiation by activating the MNK1/2/EIF4E pathway and enhancing NFATc1 protein expression. Moreover, targeting the MNK1/2 pathway diminished arthritic bone erosion. These results identified a novel role of c-FMS proteolysis in osteoclastogenesis and the pathogenesis of arthritic bone erosion.

5 cells were performed with a FACS Canto (BD Biosciences) and analyzed with FlowJo software (Tree Star Inc.).

Bone-resorption pit assays
Bone-resorption activity of osteoclasts was examined using 96-well Corning Osteo Assay Surface plates (Sigma). Mouse OCPs were plated at a seeding density of 1x10 4 per well and incubated with M-CSF (20ng/ml) and RANKL (50 ng/ml) for 5 days, with exchange of fresh M-CSF and RANKL every two days. After removing cells with 10% bleach solution, plates were stained with 1% toluidine blue solution to visualize the formation of pits. Resorbed area was analyzed using OsteoMeasure software (OsteoMetrics, Inc.).

MASS spectrophotometer assay
293T cells were transfected with pCMV6-Entry-c-FMS-MYC-DDK (NM_005211, Origene, Rockville, MD) using Lipofectamin 3000 (Thermofisher scientific). Transfected cells were incubated with M-CSF (20ng/ml) for one day and nuclear proteins were immunoprecipitated (IP) with antibodies against N-terminal of c-FMS (Santa Cruz; H-300 and R&D systems; clone #61780) to remove full-length c-FMS as a negative selection. Subsequently, the IP-proteins were incubated with either mouse IgG or DDK-tag Ab conjugated magnetic bead (Origene). Proteins bound to ab-beads were eluted with water. Samples were subjected to SDS PAGE gel was submitted for the mass spectrophotometery assay. Mass Spectrometry assay (n=2) was performed by The Taplin Biological Mass Spectrometry Facility in Harvard Medical School. Briefly, excised gel bands were cut into approximately 1 mm 3 pieces. Gel pieces were then subjected to a modified in-gel trypsin digestion procedure 8 . Gel pieces were washed and dehydrated with acetonitrile for 10 min. followed by removal of acetonitrile. Pieces were then completely dried in a speed-vac. Rehydration of the gel pieces was with 50 mM ammonium bicarbonate solution containing 12.5 ng/µl modified sequencing-grade trypsin (Promega, Madison, WI) at 4ºC. After 45 min., the excess trypsin solution was removed and replaced with 50 mM ammonium bicarbonate solution to just cover the gel pieces. Samples were then placed in a 37ºC room overnight. Peptides were later extracted by removing the ammonium bicarbonate solution, followed by one wash with a solution containing 50% acetonitrile and 1% formic acid. The extracts were then dried in a speed-vac (~1 hr). The samples were reconstituted in 5 -10 µl of HPLC solvent A (2.5% acetonitrile, 0.1% formic acid). A nano-scale reverse-phase HPLC capillary column was created by packing 2.6 µm C18 spherical silica beads into a fused silica capillary (100 µm inner diameter x ~30 cm length) with a flame-drawn tip 9 . After equilibrating the column each sample was loaded via a Famos auto sampler (LC Packings, San Francisco CA) onto the column. A gradient was formed and peptides were eluted with increasing concentrations of solvent B (97.5% acetonitrile, 0.1% formic acid). As peptides eluted they were subjected to electrospray ionization and then entered into an LTQ Orbitrap Velos Pro ion-trap mass spectrometer (Thermo Fisher Scientific, Waltham, MA). Peptides were detected, isolated, and fragmented to produce a tandem mass spectrum of specific fragment ions for each peptide. Peptide sequences (and hence protein identity) were determined by matching protein databases with the acquired fragmentation pattern by the software program, Sequest (Thermo Fisher Scientific, Waltham, MA) 10 . All databases include a reversed version of all the sequences and the data was filtered to between a one and two percent peptide false discovery rate.

6
The Ingenuity Pathway Analysis (IPA) IPA was used to analyze the functions of FICD-interacting proteins obtained from mass spectrophotometry. The molecular and cellular function was used to predict the functions whose change in enrichment relative to control could explain the interaction with FICDs.

Immunocytochemistry
Human CD14 + -monocytes were cultured with M-CSF (20ng/ml) in culture slide (BD Falcon;REF 354104) for 2days. Cells were fixed with 3.7% formalin in PBS for 20 min at room temperature. Cells were permeabilized with 1% triton X-100 for 5min, washed 3 times before blocking with solution that contains 5% horse serum, 5% Goat and 1% BSA (without IgG) in PBS for 1h. Cells were incubated with primary antibody C-terminus specific c-FMS Ab (SantaCruz Biotechnology; sc-692) overnight at 4°C followed by incubation with anti-rabbit Alexa Fluor 488-conjugated secondary antibody (A11008, Thermo Fisher Scientific) for 40 min in room temperature. After washed, finally cells were mounted with ProLongTMGold antifade regent with-DAPI (P36931, Invitrogen). The stained cells were imaged using a Zeiss Axioplan microscope (Zeiss) with an attached Leica DC 200 digital camera (Leica) or a confocal microscope system (Zeiss LSM 880, Laser excitation/emission: 405/425 and 488/525). To determine c-FMS in the nucleus, confocal three-dimensional Z-stacks were acquired for each sample using a a Plan-Apochromat 63 × /1.4 oil Dic M27 objective (Zeiss, Germany) with a slice of increment of 0.5 μm. The images were processed with Image j-Fiji software.
Immunoprecipitation 2 x10 6 BMDMs from LysM cre/cre mice were seeded into 100mm dish and were incubated with M-CSF (10ng/ml) for overnight. Cells were treated with 50ng/ml RANKL for one additional day and lysed with RIPA buffer with proteinase inhibitor cocktail. An equal amount of cell lysates were incubated with magnetic beads conjugated with anti HA-Tag antibody (Thermo Fisher scientific; 88836) for 24h at 4 °C. The beads were washed 5 times with washing buffer (20 mM HEPES [pH 7.5], 150 mM NaCl, 0.1% NP-40, 1% glycerol, protease and phosphatase inhibitors). Proteins eluted from the bead with elution buffer (pH 2.8, Prod#1858606). The sample were incubated in 95 °C for 10 mins and then were analyzed by immunoblotting.
Micro-CT and histomorphometry analysis μ-CT analysis 11 was performed as described previously 12 , and all samples were included in the analysis conducted in a blinded manner. For μCT analysis, prior to decalcification, femurs with intact joints were scanned by microCT, with an isotropic voxel resolution of 6 µm (μCT35, Scanco, Bruttisellen, Switzerland; 55kVp, 145μA, 600ms integration time) to evaluate morphological changes in bone. Bone morphology in the femur was examined in two regions: the diaphysis and the metaphysis. For cortical bone, the volume of interest (VOI) encompassed cortical bone within a 231-slice section in the diaphysis. For trabecular bone, the VOI encompassed a 200-slice section in the metaphysis, proximal to the growth plate. To ensure exclusion of primary spongiosa in the growth plate, VOIs began 50 slices proximal to the median of the growth plate. Representative trabecular images were composed of the 100 slices either proximal or distal to the growth plate. Outcome parameters for cortical bone included thickness and tissue mineral density (TMD). Trabecular bone parameters included bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and trabecular TMD. 3D reconstructions were generated by stacking thresholded 2D images from the contoured region.
Histomorphometry experiment was performed with tarsal bone of vehicle or MDL28170 treated mice. Bone histomorphometric analysis was performed in a blinded, nonbiased manner using a computerized semi-automated system (Osteomeasure, TN) with light microscopy. The tarsal bones were fixed in 4% paraformaldehyde for 2 days, were decalcified with 10% neutral buffered EDTA (Sigma-Aldrich), and were embedded in a paraffin. The quantification of osteoclast was performed in paraffin embedded tissues that were stained for TRAP and Methyl green (Vector Laboratories). Osteoclast cells 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 13 .

Bone formation assay
To assess dynamic histomorphometry, mice received intraperitoneal injections of 10 mg/kg of calcein, 5 days and 2 days before death. Bones were fixed for one day in 4% paraformaldehyde. After decalcification for one day, bones were incubated in 30 % sucrose overnight and subsequently embedded in OCT compound (Thermo Fischer Scientific, Waltham, MA). Mineral apposition rate (MAR) and Bone formation rate (BFR) were analyzed by a computerized semiautomated system (Osteomeasure, TN) with fluorescent microscopy.

K/BXN serum transfer arthritis model
For arthritis experiments, K/BxN serum pools were prepared as described previously 14 . Arthritis in 8-week-old C57BL/6J male mice (The Jackson Laboratory) was induced by intraperitoneal injection of 100 µl of K/BxN serum on days 0 and 2. . To analyze the effect of MDL28170 and CGP57380, the mice were randomized and treated with either vehicle (n=10), MDL28170 (10 mg/kg) or CGP57380 (40mg/kg) with intraperitoneally (i.p) every day for 11 or 13 days. Vehicle or MDL28170 were prepared in 2.5% DMSO and 10% KLEPTOSE pH7.0 (Roquette Phama). CGP57380 was prepared in 4% DMSO and 30% PEG300 (Selleckchem) in 0.9% saline solution (BD science) 15 . The development of arthritis was monitored by measuring the thickness of wrist and ankle joints using dial-type calipers (Bel-Art Products) and scoring the wrist and ankle joints. For each animal, joint thickness was calculated as the sum of the measurements of both wrists and both ankles. Joint thickness was represented as the average for every treatment group. The severity of arthritis was scored in a blinded fashion by four investigators for each paw on a 3point scale, in which 0 = normal appearance, 1 = localized edema or erythema over one surface of the paw, 2 = edema or erythema involving more than one surface of the paw, 3 = marked edema or erythema involving the whole paw. The scores of all four paws were added for a composite score 16 .

Supplemental table 2. Primers and Oligonucleotides
A list of primers used in this study