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Keywords:

  • OSTEOCLAST;
  • BIOSYNTHESIS;
  • BIOENERGETICS;
  • GLYCOLYSIS;
  • GLUTAMINOLYSIS

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References
  10. Supporting Information

The osteoclast is a giant cell that resorbs calcified matrix by secreting acids and collagenolytic enzymes. The molecular mechanisms underlying metabolic adaptation to the increased biomass and energetic demands of osteoclastic bone resorption remain elusive. Here we show that during osteoclastogenesis the expression of both glucose transporter 1 (Glut1) and glycolytic genes is increased, whereas the knockdown of hypoxia-inducible factor 1-alpha (Hif1α), as well as glucose deprivation, inhibits the bone-resorbing function of osteoclasts, along with a suppression of Glut1 and glycolytic gene expression. Furthermore, the expression of the glutamine transporter solute carrier family 1 (neutral amino acid transporter), member 5 (Slc1a5) and glutaminase 1 was increased early in differentiation, and a depletion of L-glutamine or pharmacological inhibition of the Slc1a5 transporter suppressed osteoclast differentiation and function. Inhibition of c-Myc function abrogated osteoclast differentiation and function, along with a suppression of Slc1a5 and glutaminase 1 gene expression. Genetic and pharmacological inhibition of mammalian target of rapamycin (mTOR), as well as the activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK), inhibited osteoclastogenesis. Thus, the uptake of glucose and glutamine and utilization of the carbon sources derived from them, coordinated by HIF1α and c-Myc, are essential for osteoclast development and bone-resorbing activity through a balanced regulation of the nutrient and energy sensors, mTOR and AMPK. © 2013 American Society for Bone and Mineral Research


Introduction

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References
  10. Supporting Information

The development of osteoclasts, bone-resorbing polykaryons that originate from hematopoietic precursors, is essential for meeting the structural and metabolic demands of the skeleton under changing mechanical conditions as well as the nutritional environment imposed on the organism. Osteoclasts play key roles in the regulation of bone mass as well as quality; a deficiency of osteoclasts results in an increased but fragile bone state known as osteopetrosis, while excessive osteoclastic activity underlies the decreased bone mass and fragility fractures that are hallmarks of osteoporosis. Unraveling the molecular program that drives the differentiation and function of osteoclasts during the lifelong continuous cycle of bone remodeling is important for a better understanding of the pathogenesis of metabolic bone disease as well as the intricate mechanisms controlling the volume and strength of the skeleton that enables the fundamental activities of daily life.

The skeleton is constantly being remodeled, and each remodeling cycle starts with the bone resorption activity performed by osteoclasts.[1, 2] Osteoclasts are large, multinucleated cells of hematopoietic origin that specialize in bone resorption.[3, 4] Osteoclastogenic stimulation by receptor activator of NF-κB ligand (RANKL) initiates a cascade of events that leads to the activation of a nuclear genetic program orchestrated by the transcription factors NF-κB, c-Fos/activator protein 1 (AP-1) and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) via intracellular kinases, such as I-κB kinase (IKK) and extracellular signal-regulated kinase (ERK).[5] Bone marrow macrophages (BMMs) first proliferate actively and become committed preosteoclasts that are tartrate-resistant acid phosphatase (TRAP)-positive mononuclear cells, which then fuse to one another to become multinucleated, mature osteoclasts.

Mature bone-resorbing osteoclasts adhere tightly to the bone surface at the sealing zone, and dissolve both inorganic and organic components of the bone matrix by secreting protons and collagenolytic enzymes, respectively, through the ruffled border membrane into the closed space between the membrane and bone surface, where bone degradation actually takes place.[3, 4] As such, bone resorption is an energy-demanding process, and osteoclasts are assumed to undergo metabolic adaptation during the course of differentiation so as to meet the increased demand for adenosine triphosphate (ATP). However, little is known about the metabolic profile and/or bioenergetics of osteoclasts, or how such metabolic adaptations that are required to meet the increased demand for ATP take place.

We have previously demonstrated that mitochondrial biogenesis is stimulated during osteoclast differentiation through the action of peroxisome proliferator-activated receptor-gamma coactivator 1 beta (PGC-1β), together with the uptake of the iron-transferrin complex through the upregulated transferrin receptor[6]; this contributes to the activation of mitochondrial respiration by providing iron, in the form of heme and iron-sulfur clusters, to respiratory proteins.[6] In order to further explore the energy source and define the osteoclast metabolic signature, we have examined the metabolic alterations that took place during the course of osteoclastogenesis. Here we show that osteoclast differentiation is associated with a metabolic reprogramming that is characterized by an increased expression of glucose and glutamine transporters, followed by glycolysis and glutaminolysis. This metabolic adaptation is orchestrated by two transcription factors, hypoxia-inducible factor 1-alpha (Hif1α) and c-Myc, and is dependent upon a coordinated balance between nutrient and energy sensors; ie mammalian target of rapamycin (mTOR) and adenosine monophosphate (AMP)-activated protein kinase (AMPK).

Materials and Methods

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References
  10. Supporting Information

Reagents

Recombinant murine macrophage colony-stimulating factor (M-CSF) and RANKL were expressed in our laboratory as described.[7] The anti–glucose transporter 1 (Glut1), anti–solute carrier family 1 (neutral amino acid transporter), member 5 (Slc1a5) and anti-β-actin antibodies were purchased from Santa Cruz Biotechnologies, Inc. (dilution 1:200, sc-7903; Santa Cruz, CA, USA), Cell Signaling Technology, Inc. (dilution 1:500, #5345; Danvers, MA, USA), and BioVision, Inc. (dilution 1:1000, 3598–100; Milpitas, CA, USA), respectively. The anti–phospho-phosphorylating eukaryotic initiation factor 4E binding protein 1 (4EBP1) antibody and 4EBP1 antibody were purchased from Cell Signaling Technology, Inc. (dilution 1:1000; Danvers, MA, USA). α-Modified essential medium (α-MEM) was obtained from GIBCO, Life Technologies Corp. (Carlsbad, CA, USA), α-MEM without L-glutamine from Sigma (St. Louis, MO, USA) and α-MEM without D-glucose from Cell Science & Technology Institute, Inc. (Sendai, Japan). L-γ-glutamyl-p-nitroanilide (GPNA), dimethyl-α-ketoglutarate (DM-α-KG), 10074-G5, rapamycin, and metformin were purchased from Sigma, and JQ1, Torin1, and 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) from the Cayman Chemical company (Ann Arbor, MI, USA), Toronto Research Chemicals Inc. (North York, Canada) and Enzo Life Sciences Inc. (Farmingdale, NY, USA), respectively. All other chemicals were obtained from Sigma.

Mice

Eight-week-old to 10-week-old C57BL/6 mice (Clea Japan Inc., Shizuoka, Japan) were used to isolate BMMs. The raptor flox mouse model has been described,[8] and the mTOR flox mouse will be described elsewhere (Hoshii et al., unpublished work). All experiments were performed in accordance with National Center for Geriatrics and Gerontology's ethical guidelines for animal care, and the experimental protocols were approved by the animal care committee.

Isolation of BMMs and cell culture

BMMs were prepared from the bone marrow of wild-type or mutant mice and cultured as described.[7] In brief, after bone marrow cells were cultured in the presence of M-CSF for 3 days, adherent cells were collected and stored in liquid nitrogen. For the experiments, BMMs were thawed and cultured with M-CSF and RANKL in the absence or presence of the test reagents. BMMs isolated from the bone marrow of the mTOR flox/– and Raptor flox/flox mice were infected with adeno-lacZ or adeno-cre virus, and then used for the osteoclastogenesis assays.

Osteoclastogenesis and bone resorption assays

After BMMs were cultured in the presence of M-CSF, RANKL, and the test reagents, osteoclasts were identified by tartrate-resistant acid phosphatase (TRAP) staining.[7] Bone resorption pit assay was performed as described[7] with minor modifications. In brief, TRAP-positive mononuclear preosteoclasts were generated by culturing BMMs in α-MEM containing 10% fetal bovine serum (FBS), M-CSF, and RANKL for 2 days, after which they were stored in liquid nitrogen. Thawed preosteoclasts were plated on dentin slices and re-stimulated with M-CSF and RANKL for an additional day; after confirmation of maturation, test reagents were added, and the resorption pits on the dentin slices were visualized after 40 hours by staining with Coomassie brilliant blue (CBB) solution and observed under light microscopy (Leica M420 Microscope, Leica Microsystems, Wetzlar, Germany). To quantify the resorption areas, six areas of the dentin slices were randomly selected and the areas of the stained resorption lacunae were determined with the NIH Image program (http://rsb.info.nih.gov/nih-image/).

RNA isolation, RT-PCR, and microarray analysis

Total RNAs were extracted from cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and were used for microarray and PCR analyses. The primers used for RT-PCR are summarized in Supplemental Tables S1 and S2. Real-time PCR amplifications of cDNA were performed using the power SYBR green PCR master mix on the 7300 fast real-time PCR system (Applied Biosystems, Foster City, CA, USA). Microarray analysis was performed as described,[6] using the mouse genome 430 2.0 Array GeneChip (Affymetrix, Santa Clara, CA, USA) and Affymetrix GeneChip Operating Software version 1.1, and the data were deposited in GEO (accession no. GSE45656).

Biochemical analysis

The lactate and glucose concentrations in the conditioned medium were determined using Lactate Assay Kit II and the Glucose Assay Kit (BioVision), respectively. Total cellular DNA, RNA, and protein contents were determined using the DNA Quantity kit (Primarycell, Hokkaido, Japan), RNeasy Micro Kit (Qiagen, Venlo, the Netherlands), and Pierce BCA Protein Assay kit (Thermo Scientific, Rockford, IL, USA).

Immunoblotting

After cells had been washed and lysed, cell lysates were boiled in SDS sample buffer and subjected to electrophoresis on 10% SDS-PAGE. Proteins were transferred to nitrocellulose membranes using a semi-dry blotter (Bio-Rad Laboratories, Hercules, CA, USA) and incubated in blocking solution (5% nonfat dry milk in TBS containing 0.1% Tween 20) for 1 hour to reduce nonspecific binding. Membranes were then exposed to primary antibodies overnight at 4°C, washed three times, and incubated with secondary goat anti-mouse or rabbit immunoglobulin G (IgG) horseradish peroxidase-conjugated antibody for 1 hour. Membranes were washed extensively, and enhanced chemiluminescence detection assay was performed according to the manufacturer's directions.

RNA interference

To generate lentiviral particles, 293T cells were transfected with the MISSION shRNA plasmid DNA and MISSION Lentiviral Packaging Mix (Sigma) using X-tremeGENE 9 DNA Transfection Reagent (Roche Applied Science, Penzberg, Upper Bavaria, Germany), and the supernatant was collected in a period of 24 to 48 hours. For infection, BMMs were incubated with lentiviral supernatants along with polybrene (4 µg/mL) for 24 hours followed by selection with puromycin (2 µg/mL) for 48 hours. The target sequences were 5′-CCGGCCAGTTACGATTGTGAAGTTACTCGAGTAACTTCACAATCGTAACTGGTTTTTG-3′ for Hif1α, and 5′-CCGGGCAGTGTTCATCGCACAACTACTCGAGTAGTTGTGCGATGAACACTGCTTTTTG-3′ for Slc1a5.

Statistical analysis

Data are expressed as the mean ± SD. Statistical analysis was performed using Student's t test. Values were considered statistically significant at p < 0.05.

Results

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References
  10. Supporting Information

Increased glycolysis during osteoclast maturation

We employed the established model of ex vivo osteoclastogenesis driven by M-CSF (25 ng/mL) and RANKL (50 ng/mL) stimulation at day 0 and day 2, which was completed in 4 days. In this model, primary mouse BMMs proliferate mainly during the first 2 days, become TRAP-positive mononuclear preosteoclasts (preOC) on day 2, and then multinucleated mature osteoclasts (mOC) on day 4. In fact, the DNA content doubled at the preOC stage (day 2), and reached a plateau thereafter when cell fusion started to take place (Supplemental Fig. S1A). RNA content, corrected for DNA content, markedly increased until day 3 and plateaued thereafter, and the protein/DNA ratio increased, especially during the latter half of the entire process (Supplemental Fig. S1A). These data suggest that osteoclast development and maturation is associated with a substantial increase in biomass, even after DNA synthesis ceases at the preOC stage; in other words, the mature osteoclast with multiple nuclei does not just increase in size as a result of cell fusion, but rather, actively increases in RNA and protein content per unit DNA. The molecular mechanism of metabolic adaptation that enables this biosynthetic and bioenergetic transformation in osteoclastogenesis remains at present unknown.

We performed global gene expression analysis with DNA microarrays during osteoclastogenesis; ie, at the three distinct stages of BMM, preOC, and mOC. BMMs constitutively expressed Glut1, known to be the primary glucose transporter in hematopoietic cells,[9] and its expression was markedly increased in mOC (Supplemental Fig. S1B). RT-PCR confirmed that the expression of Glut1 mRNA was increased toward the final maturation stage (Fig. 1A). Western blot analysis also demonstrated an increase in the GLUT1 protein in preOC and mOC compared with BMM (Fig. 1B).

image

Figure 1. Increased expression of Glut1 and the requirement of glucose in osteoclastic bone resorption. Increased Glut1 mRNA expression by RT-PCR (A) and GLUT1 protein by Western blot analysis (B) in osteoclasts. (C) Increased lactate production and glucose consumption during osteoclastogenesis. Preosteoclasts were cultured with M-CSF in the presence or absence of RANKL, and the lactate and glucose concentrations in the medium were determined at 24 hours. **p < 0.01 (n = 3). Glucose deprivation inhibits osteoclastic bone resorption (E), but not differentiation (D). In order to assess the effect of glucose on the bone-resorbing function, preosteoclasts were plated on dentin slices and re-stimulated with M-CSF and RANKL for 1 day, and after the confirmation of maturation, osteoclasts on dentin were cultured in the absence or presence of 5 mM glucose (E). Resorption pits were stained with CBB on day 3 (bottom left) and pit areas were quantified using NIH image program (right). ***p < 0.001 (n = 4).

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The expression of glycolytic genes, including hexokinase (HK), phosphofructokinase (PFK), and pyruvate kinase (PKM, muscle type), which are rate-limiting enzymes that control the pace of glycolysis and outflow from the glycolytic pathway to the citric or tricarboxylic acid (TCA) cycle, was progressively increased toward the final maturation stage of osteoclast differentiation (Supplemental Fig. S1B). The expression of lactate dehydrogenase (LDH) A and B and vascular endothelial growth factor (VEGF) A and B was also increased in mOC (Supplemental Fig. S1B and C). Accordingly, lactate secretion as well as glucose consumption increased significantly along with osteoclast differentiation (Fig. 1C). Although osteoclast differentiation was not inhibited in α-MEM that was deficient in D-glucose (Fig. 1D), glucose deprivation significantly inhibited the bone-resorbing activity, as determined by pit assays (Fig. 1E). To assess the effect of glucose on the function of mature osteoclasts in these experiments, preosteoclasts were plated on dentin slices and re-stimulated with M-CSF and RANKL, and after maturation was confirmed, the bone-resorbing activity was assessed in glucose-free α-MEM without or with supplementation with 5 mM D-glucose (Fig. 1E). Collectively, it is thus suggested that glucose utilization is important for osteoclastic bone resorption.

The expression patterns of pyruvate dehydrogenase (PDH), an enzyme that links glycolysis to the TCA cycle, as well as pyruvate dehydrogenase kinase (PDK) that inhibits PDH, and citrate synthase (CS) and isocitrate dehydrogenase (IDH), along with the recently identified mitochondrial pyruvate carrier (Mpc) 1 and 2,[10, 11] were all substantially increased during osteoclastogenesis (Supplemental Fig. S1D). Taken together with our previous report on the increased mitochondrial biogenesis mediated by PGC-1β, [6] it is reasonable to postulate that the high energy demands for generating osteoclasts are met through the TCA cycle and subsequent oxidative phosphorylation in mitochondria, coupled with increased glycolysis.

HIF1α in glucose metabolism and osteoclastic bone resorption

Osteoclasts usually reside in resorption lacunae, where oxygen tension is believed to be very low.[12] Thus, under these in vivo conditions, the HIF protein stabilized in the hypoxic microenvironment is likely to play an important role in the transcriptional activation of glut1, glycolytic enzymes, and VEGF in osteoclasts, as shown in Fig. 1 and Supplemental Fig. S1. Previously, hypoxia in the placenta was shown to affect osteoclast size through the Fos-like antigen-2 (Fra2)/leukemia inhibitory factor (LIF)/prolyl hydroxylase domain-containing protein 2 (PHD2)/HIF axis[13]; however, the pathway that regulates giant osteoclast formation through local hypoxia in the adult skeleton remains to be determined.

We assessed the direct role of HIF in osteoclast differentiation and function. Knockdown of Hif1α did not inhibit the formation of multinucleated osteoclasts, in fact it had a modest stimulatory effect on osteoclastogenesis (Fig. 2A). In contrast, knockdown of Hif1α markedly inhibited the bone-resorbing function of mOC, as determined by pit assays (Fig. 2B). The knockdown of Hif1α was associated with a decreased expression of Glut1, PFK, LDH-A, and VEGF-A in mature resorbing osteoclasts, as determined by quantitative RT-PCR (Fig. 2C), whereas it had no inhibitory effect on the expression of NFATc1, a master regulator of osteoclastogenesis, or cathepsin K, a molecular marker of osteoclast differentiation (Fig. 2C). These data are consistent with the concept that HIF1α activates the transcription of Glut1 and rate-limiting glycolytic enzymes in mature osteoclasts, thereby promoting glucose uptake and glycolysis, and ultimately regulating the bone-resorbing function.

image

Figure 2. Hif1α in the induction of glycolytic enzymes and bone resorption. (A) Knockdown of Hif1α does not inhibit osteoclast differentiation. Confirmation of decreased Hif1α expression (left), with representative TRAP-positive osteoclasts formed in the absence (mock) or presence of shRNA (right). (B) Inhibition of osteoclastic bone resorption by the knockdown of Hif1α. Resorption pits were stained with CBB (left) and pit areas were quantified using NIH image (right). ***p < 0.001 (n = 3). (C) Knockdown of Hif1α suppresses the expression of Glut1, PFK-L, LDH-A, and VEGF-A, but not NFATc1 or CathK (each corrected for β-actin). PFK-L = phosphofructokinase, liver; LDH-A = lactate dehydrogenase a; VEGF-A = vascular endothelial growth factor a; NFATc1 = nuclear factor of activated T cells c1; CathK = cathepsin K. ***p < 0.001 (n = 3).

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L-glutamine is required for osteoclastogenesis

Although the ex vivo osteoclastogenesis assay that uses M-CSF and soluble RANKL is widely applied, it is recognized that the process is sensitive to various factors, such as the culture medium, serum lot, and cell density. In fact, we found that a certain α-MEM product was ineffective in generating osteoclasts even with the same lot of serum and M-CSF/RANKL (Fig. 3A). Comparison of the constituents in the medium revealed that the osteoclastogenesis-incompetent α-MEM lacked L-glutamine, even though α-MEM usually contains 2 mM L-glutamine. In fact, when L-glutamine was added to the osteoclastogenesis-defective α-MEM in increasing concentrations, osteoclast formation was restored in a dose-dependent manner, with the maximal effect being observed at 1.5 to 2.0 mM L-glutamine (Fig. 3A, and data not shown). When L-glutamine was added at different time points during the 4-day differentiation process, supplementation of L-glutamine either during the first or the second half period significantly restored osteoclast formation, with the restoring effect slightly larger in the latter case (Supplemental Fig. S2A, and data not shown). These data suggest that although L-glutamine seems to be required from an early phase of osteoclast differentiation, it may have more significant impact in a later maturation stage.

image

Figure 3. Glutamine in osteoclast differentiation. (A) Supplementation of L-glutamine to glutamine-deficient α-MEM medium restores osteoclast development. Representative TRAP-positive osteoclasts formed in the absence or presence of L-glutamine (–/+ Gln) are shown (left) with quantitation of the number of TRAP-positive multinucleated cells (more than three nuclei) per well in a 96-well plate (right). ***p < 0.001 (n = 3). Expression of Slc1a5 mRNA (B) and the SLC1a5 protein (C) during osteoclastogenesis. (D) Inhibition of osteoclastogenesis by GPNA, an inhibitor of glutamine transporter ***p < 0.001 (n = 3). (E) Knockdown of Slc1a5 inhibits the formation of osteoclasts. ***p < 0.001 (n = 3). (F) Exogenous dimethyl-α-ketoglutarate (DM-α-KG) rescues the suppressive effect of glutamine deficiency on osteoclast formation. A membrane-permeable α-KG analog, DM-α-KG, was added to glutamine-deficient α-MEM medium, and the number of TRAP-positive multinucleated cells per well in a 96-well plate was counted. ***p < 0.001 (n = 3).

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Inhibition of SLC1A5 suppresses osteoclastogenesis

The gene expression analysis revealed that the expression of Slc1a5, a high-affinity, Na+-dependent transporter of L-glutamine,[14, 15] was markedly elevated at the transition from BMM to preOC and stayed at a high level in mOC (Supplemental Fig. S2B). This was confirmed by RT-PCR (Fig. 3B) and Western blot analysis (Fig. 3C). In order to see whether SLC1A5 functions in osteoclastogenesis, the effect on osteoclast formation of GPNA, an inhibitor of SLC1A5-regulated transport,[15] was examined in ex vivo cultures. As shown in Fig. 3D, GPNA dose-dependently and markedly inhibited the formation of TRAP-positive osteoclasts, with a clear-cut inhibition being observed at a dose as low as 0.1 mM. This was further supported by the finding that the knockdown of Slc1a5 markedly inhibited the formation of osteoclasts (Fig. 3E). When the L-glutamine-sufficient cultures were treated with GPNA for various time periods, it turned out that the presence of GPNA on the first 2 days had a minor effect, whereas treatment on the latter 2 days exerted a substantial inhibitory effect on osteoclast formation (Supplemental Fig. S2C), suggesting that L-glutamine uptake through SLC1A5 is more important during the latter half of osteoclast differentiation, and also that the inhibitory effect of GPNA is reversible.

Glutaminolysis and anaplerosis

Microarray analysis revealed that although glutaminase 2 (Gls2, the liver type) was not expressed, Gls1, specifically transcript variant 1 (the kidney type), which converts glutamine to glutamate, was upregulated more than fivefold during osteoclastogenesis (Supplemental Fig. S2B, and data not shown). The expression of Gls1 in mOC was confirmed by RT-PCR (Supplemental Fig. S2D). These data suggest that following glutamine uptake through Slc1a5, osteoclasts are able to convert glutamine to glutamate and then to α-ketoglutarate (α-KG).

The importance of an α-KG supply through glutaminolysis in osteoclastogenesis was underscored by the finding that the inhibition of osteoclast formation by glutamine deprivation was rescued by the addition of a membrane-permeable α-KG analog, dimethyl-α-KG (DM-α-KG) in a dose-dependent manner (Fig. 3F). Thus, glutamine uptake, glutaminolysis and a supply of α-KG to the TCA cycle as an anaplerotic substrate plays an important role in osteoclast development. This is a very similar situation to the metabolic reprogramming reportedly characterized in cancer cells.[16]

c-Myc in induction of Slc1a5 and glutaminolysis

In order to gain further insight into the transcriptional network that underlies the metabolic adaptation of osteoclasts, we assessed the role of c-Myc in osteoclast differentiation and function. In order to target c-Myc transcription function, we first used the recently developed cell-permeable small molecule JQ1, which displaces bromodomain and extra-terminal (BET) proteins from chromatin by competitively binding to the acetyl-lysine recognition pocket of BET bromodomains, thereby downregulating Myc transcription.[17, 18] As shown in Fig. 4A, B, JQ1 suppressed the osteoclast differentiation (Fig. 4A) as well as bone-resorbing function of mature osteoclasts dose-dependently (Fig. 4B). Regarding the timing of JQ1 activity during osteoclastogenesis, its presence in the latter half had a slightly greater inhibitory effect (Supplemental Fig. S3A), suggesting that c-Myc function is more important in this period. It was evident that the inhibition of c-Myc with JQ1 significantly decreased the expression of Slc1a5 and Gls1 in mature osteoclasts (Fig. 4C), which is consistent with a previous report demonstrating that c-Myc enhances glutaminase as well as glutamine transporter expression and glutamine metabolism.[19] 10074-G5, another inhibitor of c-Myc that acts by inhibiting c-Myc/Max interaction,[20] also inhibited the differentiation as well as bone-resorbing activity of mature osteoclasts dose-dependently (Supplemental Fig. S3B). Taken together, the data are consistent with the concept that c-Myc activates the transcription of Slc1a5 and glutaminase, promoting glutamine uptake and glutaminolysis, and as a result contributing to osteoclast differentiation and function.

image

Figure 4. c-Myc and mTOR in osteoclast differentiation and function. (A) Inhibition of c-Myc function with JQ1 suppresses osteoclast differentiation. Representative TRAP-positive osteoclasts formed in the absence or presence of JQ1 are shown (top) along with the number of TRAP-positive multinucleated cells per well in a 96 well plate (bottom). ***p < 0.001 (n = 3). (B) Inhibition of the bone-resorbing function of mature osteoclasts by JQ1. ***p < 0.001 (n = 3). (C) Inhibition of c-Myc function with JQ1 suppresses the expression of Slc1a5 and Gls1 (corrected for β-actin) in mature osteoclasts by quantitative RT-PCR. ***p < 0.001 (n = 3). Inhibition of mTOR with Torin1 suppresses osteoclastogenesis (D) and the bone-resorbing function (E). ***p < 0.001 (n = 3). (F) Deletion of mTOR or raptor inhibits osteoclastogenesis. BMMs isolated from mTOR flox/– or Raptor flox/flox mice were stimulated for osteoclastogenesis in the absence or presence of an adeno-cre vector, and the number of TRAP-positive multinucleated cells per well in a 96-well plate was determined. ***p < 0.001 (n = 3).

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mTOR in osteoclast differentiation and bone resorption

mTOR is an atypical protein kinase that regulates growth and metabolism by integrating information on nutrient, energy, and oxygen levels, with the drug rapamycin being known to be a partial, allosteric inhibitor of mTOR kinase activity. mTOR associates with various proteins, such as raptor and rictor, to form mTOR complex 1 (mTORC1) and mTORC2, respectively.[21, 22] Recent studies have suggested that the interaction of mTORC1 with Ras-related GTP binding proteins (Rag GTPases) (RagA or RagB and RagC or RagD in mammals) is important for its activation by amino acids.[23, 24] Osteoclasts constitutively express not only mTOR but also raptor and rictor, as well as RagA and a high level of RagC (Supplemental Fig. S4A). Further, it has been shown that amino acids induce the translocation of mTORC1 to a lysosomal compartment through the Ragulator complex, which is comprised of late endosomal/lysosomal adaptor, MAPK and MTOR activator 3 (MAPKSP1) (MP1), late endosomal/lysosomal adaptor, MAPK and MTOR activator 2 (ROBLD3) (p14), and late endosomal/lysosomal adaptor, MAPK and MTOR activator 1 (Lamtor1).[25] All of these three components of the Ragulator complex are expressed in osteoclasts (Supplemental Fig. S4A).

Because the data so far have suggested that energy status and nutrients, especially glucose and glutamine, are important regulators of osteoclastic development and/or bone resorption, we assessed the involvement of mTOR in osteoclastogenesis, first by treating osteoclastogenic cultures with various doses of rapamycin. As shown in Supplemental Fig. S4B, treatment with 100 nM rapamycin significantly inhibited the formation of TRAP-positive multinucleated osteoclasts. Although it had a more potent effect when added during the first half of differentiation, its presence during the latter half also exhibited a dose-dependent and significant inhibition (Supplemental Fig. S4B). On the other hand, rapamycin had little effect on the bone resorbing function of mature osteoclasts (Supplemental Fig. S4C).

Because rapamycin did not display any drastic effect on the bone-resorbing activity, we reasoned that it was not sufficient for full inhibition of mTOR in osteoclasts, and utilized a more potent, catalytic site ATP-competitive mTOR inhibitor, Torin1.[26] Indeed, Torin 1 inhibited mTOR activity whereas rapamycin had little effect (Supplemental Fig. S4D). Torin1 potently inhibited osteoclast differentiation at 10 nM (Fig. 4D); again, its effect was slightly more prominent when added during the first half of the differentiation process (Supplemental Fig. S4E). Torin1 also potently and dose-dependently inhibited the bone-resorbing function of mature osteoclasts (Fig. 4E). We also examined the role of mTOR in osteoclastogenesis using a genetic approach. BMMs were isolated from the bone marrow of mTOR flox/– or raptor flox/flox mice and treated with an adeno-cre vector ex vivo to knock down mTOR or raptor, respectively (Supplemental Fig. S5A), and their differentiation capacity was assessed. As shown in Fig. 4F and Supplemental Fig. S5B, deletion of mTOR or raptor significantly inhibited the formation of TRAP-positive osteoclasts. Because raptor is an essential component of mTORC1 and its depletion had almost the same suppressive effect on osteoclastogenesis as the deletion of mTOR, it is suggested that mTOR, more specifically mTORC1, plays a dominant role in osteoclast development as a nutrient sensor. When AMPK, a sensor of increased AMP and adenosine diphosphate (ADP), was activated pharmacologically by either AICAR or metformin, osteoclastogenesis was significantly inhibited (Supplemental Fig. S5C), suggesting that ATP has to be maintained above a certain threshold to prevent the accumulation of AMP/ADP and activation of AMPK.[27]

Discussion

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References
  10. Supporting Information

This study has revealed a complex metabolic network integrated with the osteoclast differentiation program. A metabolic shift to aerobic glycolysis in certain cancers is known as the Warburg effect.[16, 28] It is also known that during T-cell activation, an antigen-specific signal through T cell receptor (TCR) and a costimulatory signal through CD28 cooperate to facilitate the metabolic conversion of T cells from quiescence to proliferation.[29] This study demonstrates glucose and glutamine are essential carbon sources not only for osteoclast differentiation, but also for the acquisition of bone-resorbing activity in mature, multinucleated osteoclasts, an effect which is orchestrated by two transcription factors, HIF-1α and c-Myc (Supplemental Fig. S6).

HIF-1α has been shown to play major roles in skeletal biology, first in chondrocytes[30] and then in osteoblasts.[31] Hypoxia has been shown to stimulate osteoclast formation and resorption,[32] locally through HIF-1α[33] as well as systemically as a result of HIF-1α activation in the placenta and through the molecular relays of Fra2/LIF/PHD2/HIF.[13] It was demonstrated in the present study that HIF-1α knockdown does not suppress osteoclast differentiation, but does markedly inhibit the bone-resorbing activity of mature osteoclasts. Further, our data suggest that HIF-1α functions through the stimulation of genes involved in glucose transport and glycolysis, because HIF-1α knockdown is associated with a repression of Glut1 and glycolytic genes. The stimulatory effect of D-glucose (5 mM) on bone resorption in the current study is consistent with a previous report showing that glucose supports bone resorption activity in isolated chicken osteoclasts, although a much higher concentration (25 mM) was used in that study.[34] In fact, an inhibitory effect of a high glucose concentration (40 mM) on the number of TRAP-positive osteoclasts in mouse bone marrow cultures has been reported,[35] and the optimal glucose level for osteoclast development and its relation to local oxygen tension in vivo remain to be determined.

This is the first report, to our knowledge, that L-glutamine is essential not only for osteoclastogenesis but also for the bone-resorbing activity, and we have provided evidence of a mechanistic link between c-Myc and glutamine metabolism in osteoclasts through the activation of SLC1a5 and glutaminas, a glutamine transporter and an enzyme that converts glutamine to glutamate. There are only a few and conflicting reports on the function of c-Myc in osteoclasts; a dominant negative Myc blocked RANKL-induced osteoclast formation in a RAW 264.7 cell line,[36] whereas c-Myc repressed the transcription of the representative osteoclast marker gene, TRAP, in a murine macrophage cell line.[37] The specific physiologic functions of c-Myc in osteoclast biology and bone metabolism in vivo remain to be addressed.

The present study has provided genetic as well as pharmacological evidence for the importance of mTOR in osteoclastogenesis. There is a report showing that mTOR is involved in the regulation of Bim expression by M-CSF and that knockdown of mTOR by RNA interference (RNAi)-induced apoptosis,[38] and inhibition of mTOR with rapamycin has been demonstrated in vitro to inhibit bone resorption[39] as well as osteoclast formation through an effect on the CCAAT/enhancer binding protein β (C/EBPβ) isoform ratio and v-maf musculoaponeurotic fibrosarcoma oncogene homolog B (MafB).[40] Regarding the function of mTOR in vivo, it has been shown that treatment with everolimus or rapamycin prevents the bone loss induced by ovariectomy (OVX) in Wistar rats[41] as well as joint destruction in a tumor necrosis factor (TNF) transgenic mouse model of rheumatoid arthritis (RA),[42] respectively. The pharmacological inhibition of mTOR with the recently developed potent mTOR inhibitor Torin1 in the current study supports these previous findings. Further, our finding that genetic deletion of mTOR or raptor (a TORC1-specific component) results in a similar level of inhibition of osteoclast differentiation points to a dominant role for mTORC1 in osteoclasts. Finally, the contrasting effects of anabolic mTOR and catabolic AMPK on osteoclast formation suggest that a balanced activity of these critical nutrients and energy sensor kinases is an important determinant of osteoclast development.

In conclusion, we have elucidated a regulatory mechanism for the osteoclast to adapt its cellular metabolism to meet the bioenergetic and biosynthetic needs related to its bone-resorbing function. The results suggest that enhanced glucose/glutamine uptake and glycolysis/glutaminolysis are hallmarks of bone-resorbing osteoclasts, and may offer novel opportunities for therapeutic intervention in metabolic bone diseases caused by osteoclastic dysfunction.

Acknowledgments

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References
  10. Supporting Information

This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas (22118007 to K. Ikeda) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Authors' roles: Study design: YI and K Ikeda. Data acquisition: YI, ST, K Ishii, and HA. Generation of mouse models: TH and AH. Data analysis and interpretation: YI, ST, K Ishii, TH, HA, AH, and K Ikeda. Drafting manuscript: YI, ST, and K Ikeda. Approving final version of manuscript: YI, ST, K Ishii, TH, HA, AH, and K Ikeda. K Ikeda takes responsibility for the integrity of the data analysis.

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  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References
  10. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
jbmr1976-sm-0001-SuppData-S1.pdf4215K

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

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