Fig. S1. Increased cellular biomass and increased expression of Glut1 and glycolytic genes with osteoclastogenesis. (A) Increased cellular RNA and protein content with osteoclastogenesis. Cells were collected on the indicated days after the initiation of osteoclast differentiation, and the cellular DNA, RNA and protein content was determined. The RNA and protein content was corrected for DNA. Day 0, 2 and 4 correspond to bone marrow macrophages (BMM), TRAP-positive mononuclear pre-osteoclasts (preOC) and multinucleated mature osteoclasts (mOC), respectively. (B-D) Results of microarray analysis in the course of osteoclastogenesis. RNA was isolated from BMM, preOC and mOC, and subjected to gene expression analysis using Affymetrix Gene Chip. HK: hexokinase, PFK: phosphofructokinase, LDH: lactate dehydrogenase, GPI: glucose phosphate isomerase, PGM: phosphoglycerate mutase, PKM: pyruvate kinase, muscle type, VEGF: vascular endothelial growth factor, PDH, pyruvate dehydrogenase, PDK: pyruvate dehydrogenase kinase, CS: citrate synthase, IDH: isocitrate dehydrogenase, Mpc: mitochondrial pyruvate carrier, Acly: ATP citrate lyase, MDH: malate dehydrogenase.
Fig. S2. L-Glutamine in osteoclastogenesis. (A) The effects of L-glutamine in a 4-day differentiation process. Osoteoclastogenic assays were performed in L-glutamine (Gln) sufficient medium (a), or glutamine-deficient medium during the second (b) or first half (c) of the culture. Representative TRAP-positive osteoclasts formed in the absence or presence of L-glutamine (−/+ Gln) are shown (bottom) along with the number of TRAP-positive multinucleated cells (more than 3 nuclei) per well in a 96 well plate (right). **p < 0.01 (n = 3) (B) Increased expression of the glutamine transporter Slc1a5 and glutaminase (Gls) 1 by microarray analysis. (C) The effects of GPNA, an inhibitor of Slc1a5-regulated transport, on osteoclastogenesis. GPNA was added for the first (a) or second half (b) of the culture, and the number of TRAP-positive multinucleated cells per well in a 96 well plate was counted (bottom). ***p < 0.001 (n = 3) (D) Increased expression of glutaminase (Gls) 1, the kidney type, in osteoclasts by RT-PCR.
Fig. S3. c-Myc in osteoclast differentiation and function. (A) The effects of JQ1, an inhibitor of c-Myc expression, on osteoclastogenesis. JQ1 was added at the indicated doses for the first (a) or second half (b) of the culture, and the number of TRAP-positive multinucleated cells per well in a 96 well plate was counted. **p < 0.01, ***p < 0.001 (n = 4) (B) 10074-G5, an inhibitor of c-myc function, dose-dependently suppresses the formation of TRAP+ osteoclasts (upper panel) as well as the bone-resorbing function (lower panel).
Fig. S4. Pharmacological inhibition of mTOR suppresses osteoclast differentiation. (A) Expression of mTOR and mTORC/Ragulator components during osteoclast differentiation by microarray analysis. (B) The effects of rapamycin on osteoclast differentiation. Rapamycin was added at the indicated doses throughout the 4-day differentiation process (a), or during the first (b) or second half (c) of the culture. Representative TRAP-positive osteoclasts formed in the absence or presence of rapamycin are shown (bottom left) along with the number of TRAP-positive multinucleated cells per well in a 96 well plate (right). ***p < 0.001 (n = 4) (C) The effects of rapamycin on osteoclastic bone resorption. (D) Osteoclasts were treated with rapamycin (100 nM), Torin1 (50 nM), or the vehicles (ethanol for rapamycin and DMSO for Torin1) for 24 hours, and mTOR activity was assessed by phosphorylation of 4EBP1. (E) The effects of Torin1 on osteoclast differentiation. Torin1 was added at the indicated doses during the first (a) or second half (b) of the culture. Representative TRAP-positive osteoclasts formed in the absence or presence of Torin1 are shown (bottom left) along with the number of TRAP-positive multinucleated cells per well in a 96 well plate (right). ***p < 0.001 (n = 4).
Fig. S5. Genetic deletion of mTOR or raptor suppresses osteoclast differentiation. (A,B) The effects of mTOR or raptor deletion on osteoclastogenesis. The mTOR or raptor gene was deleted in BMMs isolated from mTOR flox/- or Raptor flox/flox mice, respectively, by infection with an adno-cre vector (A), and the effects on TRAP+ osteoclast formation were assessed (B). (C) The effects of AMPK activation on osteoclastogenesis. AMPK was stimulated with AICAR or metformin at the indicated doses, and the number of TRAP+ osteoclasts per well in a 96 well plate was determined. ***p < 0.001, **p < 0.01 (n = 3).
Fig. S6. Schematic representation showing that in addition to the activation of the established transcription factors, NF-κB, c-Fos and NFATc1 following RANKL stimulation, HIF1α and c-MYC are required to induce the glucose and glutamine transporters, Glut1 and Slc1a5, and stimulate glycolysis and glutaminolysis, respectively, in order to coordinate the tremendous increase in biomass associated with osteoclast differentiation and to meet the bioenergetic demands for bone resorption. Thus, osteoclastic differentiation and function are assured by the maintenance of a high ATP/AMP ratio (generated through an activated TCA cycle and oxidative phosphorylation [OxyPhos] in mitochondria [Mito]), with a resulting activation of mTOR signaling and inhibition of AMPK.
Table S1. Oligonucleotide primers for PCR
Table S2. Oligonucleotide primers for q-PCR