Osteoclasts

L. Bonewald (University of Missouri at Kansas City, USA) provided the murine MLO-A5 in 1997 and MLO-Y4 in 2001 osteocyte-like cells (14 (link), 15 (link)). JJN3, 5TGM1 and MM1.S MM cell lines were provided by N. Giuliani (University of Parma, Italy) in 2006, B. Oyajobi (University of Texas at San Antonio, USA) in 2007, and S. Rosen (Northwestern University, USA) in 2003 (16 (link)–18 (link)). R. Jilka (University of Arkansas for Medical Sciences, USA) provided the OB-6 osteoblast-like cells in 1997 (19 (link)). Non-adherent osteoclast precursors were collected as described before (20 (link)). After informed consent, CD138⁺ cells from MM patients were prepared as previously detailed (16 (link)). Studies were approved by the Indiana University School of Medicine Institutional Review Board. Cell lines were authenticated by morphology, gene expression profile, and tumorigenic capacity (MM cells). Co-cultures were established by 1) adding MM cells on top of osteocyte-like cells, 2) adding MM cells in transwell chambers in a 1:5 ratio (osteocytic:MM), or 3) adding 50% conditioned media (CM) from 48h-culture of MM cells to osteocytes. DEVD (50nM), anti-TNFα (0.3μg/mL) or GSIXX (2.5–10μM) were added 1h before addition of MM cells or CM. MLO-A5 cells were treated with 0.01ng/mL TNFα, 5ng/mL TGFβ or 10ng/mL interleukin 6 (IL-6) for 4–24h. For Notch activation, MLO-A5 cells were cultured on DLL1-IgG2 or control IgG2-coated plates for 24h. For OB-6 osteoblast-like cell differentiation, cells were cultured with osteogenic media (OM; 0.2 mM ascorbic acid, 10 mM β-glycerophosphate) or OM containing 50% of CM from MLO-A5 or JJN3 cells cultured alone, or from JJN3 directly co-cultured with MLO-A5 cells.

This study was performed to evaluate the relative roles of Mmp9 and Mmp14 in regulating osteoclast function and the physiological and pathological consequences of their targeting on bone homeostasis. These objectives were addressed by (i) determining the MMP profile selectively expressed in mouse osteoclasts in vitro and in vivo, (ii) characterizing the cooperative role of Mmp9/Mmp14 in controlling bone resorption and type I collagenolysis by mouse osteoclasts in vitro, (iii) demonstrating the utility of dual MMP9 and MMP14 blockade in regulating human osteoclast-dependent bone resorption, and (iv) defining the cooperative role of Mmp9/Mmp14 in controlling mouse osteoclast-dependent bone resorption during physiologic versus pathologic bone resorption in vivo. All data presented here have been replicated in independent cohorts of three or more mice or in three or more biological replicates for in vitro experiments. Samples were assigned randomly to the experimental groups. Data collection for each experiment is detailed in the respective figures, figure legends, and methods. For genotyping primers and quantitative real-time PCR primers, see tables S1 and S2, respectively. Individual subject-level data for experiments where n < 20 are included in data file S1. Full images of Western blots are presented in data file S2.

Tibiae from virgin, lactating, and post-lactation mice (n=3 different mice in each group yielding 9 gene arrays) were processed immediately after sacrifice. Osteocyte RNA was extracted from tibia diaphyses after sequential digestion to remove surface cells such as osteoclasts and osteoblasts. As previously described^{(18 (link))}, soft tissue and periosteum were removed from the femurs, the epiphyses were cut off and bone marrow was removed by centrifugation. Diaphyses were submitted to three sequential incubations at 37°C with 0.2% type 1 collagenase (Sigma)/ 0.05% trypsin (Sigma) for 30 minutes, and then were washed with PBS. Then the bones were digested in 0.53 mM EDTA/ 0.05% trypsin (Cellgro, Mediatech, Inc, Manassas, VA) at 37°C for 30 min, rinsed with PBS and digested with 0.2% collagenase/ 0.05% trypsin solution at 37°C for 30 min. Finally, the diaphyses were rinsed with PBS, pulverized in liquid nitrogen, and the resulting bone powder added to Trizol reagent (Invitrogen, Carlsbad, CA). Total RNA was isolated as per the Trizol manufacturer’s instructions. The gene expression profile of the osteocytes was determined using the Affymetrix GeneChip system Mouse 430 2.0 array (Affymetrix, Santa Clara, CA). Sample amplification, labeling, hybridization and scanning were carried out by the microarray core of the Kansas University Medical Center, Kansas City, KS. Briefly, 100 ng total RNA was reversely transcribed to cDNA, subjected to T7 mediated in vitro-transcription amplification and labeling followed by hybridization to the array for 16 hours at 45°C. The array was washed and stained with streptavidin-phycoerythrin on the GeneChip® Fluidics Station 450, and scanned using the GeneChip® Scanner 3000 7G with autoloader. The RMA algorithm was applied to the raw data to generate intensity values. Gene filtering was then applied to identify significantly differentially regulated genes. Filters included: background filter and fold change filter (1.5 fold). Gene lists were analyzed using Bioconductor with one-way ANOVA Analysis. The data sets can be found under: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE23496.