Differential regulation of HIF-mediated pathways increases mitochondrial metabolism and ATP production in hypoxic osteoclasts

Inappropriate osteoclast activity instigates pathological bone loss in rheumatoid arthritis. We have investigated how osteoclasts generate sufficient ATP for the energy-intensive process of bone resorption in the hypoxic microenvironment associated with this rheumatic condition. We show that in human osteoclasts differentiated from CD14+ monocytes, hypoxia (24 h, 2% O2): (a) increases ATP production and mitochondrial electron transport chain activity (Alamar blue, O2 consumption); (b) increases glycolytic flux (glucose consumption, lactate production); and (c) increases glutamine consumption. We demonstrate that glucose, rather than glutamine, is necessary for the hypoxic increase in ATP production and also for cell survival in hypoxia. Using siRNA targeting specific isoforms of the hypoxia-inducible transcription factor HIF (HIF-1α, HIF-2α), we show that employment of selected components of the HIF-1α-mediated metabolic switch to anaerobic respiration enables osteoclasts to rapidly increase ATP production in hypoxia, while at the same time compromising long-term survival. We propose this atypical HIF-driven metabolic pathway to be an adaptive mechanism to permit rapid bone resorption in the short term while ensuring curtailment of the process in the absence of re-oxygenation. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Introduction
Osteoclasts are large, non-proliferative, multinucleated cells responsible for the resorptive component of bone remodelling. Osteoclast-mediated osteolysis is a diagnostic feature of rheumatoid arthritis (RA), contributing to progressive disability and predictive of poor prognosis [1]. Osteoclasts bind bone via αvβ3 integrin, then form an F-actin-rich seal to isolate a resorptive compartment above the osseous substrate. Resorption is initiated by active transport of protons across the boneapposing membrane by vacuolar H + ATPase, a process also supported by Na,K-ATPase, Ca-ATPase and gastric H,K-ATPase [2]. This acidifies the resorptive compartment, exposing the organic matrix to proteolytic enzymes also secreted from the osteoclast. Osteoclasts are additionally highly motile and these processes render bone resorption an energy-intensive process with a high demand for ATP [3,4].
Hypoxia is another feature of the rheumatoid joint and also a poor prognostic indicator [5]. Cellular adaptation to hypoxia generally entails switching to anaerobic metabolism; reducing ATP production to prevent accumulation of reactive oxygen species (ROS) [6]. This survival mechanism is largely mediated by the hypoxia-inducible factor (HIF-1, HIF-2) family of transcription factors. Post-translational stabilization of the α-subunit of HIF-1 and HIF-2 under hypoxia transactivates numerous downstream genes, including those mediating the switch to initiate metabolic adaptation to hypoxia and maintain energy/redox homeostasis [7].
First, HIF triggers a cytochrome c oxidase subunit switch (COX4-1 to COX4-2), which increases the efficiency of complex IV of the mitochondrial electron transport chain (ETC) with respect to the amounts of ATP and ROS produced [8]. Once this is insufficient to maintain homeostasis, HIF stimulates glucose transporter and glycolytic enzyme expression to increase glycolytic flux [9]. Third, HIF inhibits pyruvate dehydrogenase (PDH), the mitochondrial enzyme that converts pyruvate into acetyl CoA, by increasing expression of PDH kinase (PDK) which phosphorylates and inactivates PDH [10,11]. This reduces flux through the mitochondrial tricarboxylic acid (TCA) cycle and ETC and reduces accumulation of ROS. Fourth, HIF induces expression of BCL2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3), which competes with Beclin-1 for binding to Bcl-2, releasing Beclin-1 to stimulate mitochondrial autophagy and also reduce accumulation of ROS [12].
Hypoxia exerts various effects on osteoclasts. It reduces the viability of mature osteoclasts [13,14], increases osteoclast differentiation when combined with periods of re-oxygenation [13][14][15][16] and increases bone resorption in a HIF-1α-dependent manner [13][14][15][16][17]. Osteoclasts exhibit elevated expression of TCA cycle and oxidative phosphorylation enzymes [18], high rates of oxygen consumption [19] and numerous mitochondria [20], suggesting that high mitochondrial metabolic activity drives ATP production in these cells. The preferred substrate is glucose (via glycolysis) [19,21,22], although fatty acid oxidation has been implicated [23]. Given the high energy requirement for resorption, it could be presumed that increased resorption under hypoxia would increase cellular demand for ATP. However, switching to anaerobic glycolysis would not be expected to support this increased demand.
We have investigated the metabolic requirements of hypoxic osteoclasts to determine how they generate the requisite ATP for increased bone resorption. Identification of key components of this pathway might highlight possible therapeutic targets for amelioration of pathological bone resorption conditions.

Mitochondrial function
Intracellular ATP was assayed using CellTiter Glo (Promega, Southampton, UK). Mitochondrial dehydrogenase activity within the ETC was assessed using Alamar blue (AbD Serotec) and normalized to cell number using crystal violet, which stains nuclei independently of cellular metabolic status. Oxygen consumption was assessed using MitoXpress-Xtra-HS (Luxcel Biosciences, Cork, Ireland), a porphyrin-based phosphorescent oxygen-sensitive probe. 16 h prior to assay, osteoclasts were transferred into fresh culture medium or medium lacking glucose but supplemented with 1 mM pyruvate. Probe (10 µl) was added and the cells equilibrated at 20% or 2% O 2 . The assay was read using a FLUOstar Omega plate reader with ACU (BMG Labtech, Aylesbury, UK), held at 20% or 2% O 2 , the maximal rate of oxygen consumption being proportional to the change in probe fluorescence during the linear phase of the assay [24] (see Supplementary material, additional methodology for O 2 consumption assay). To assess relative mitochondrial number, nonyl-acridine orange was applied to cells in 5% FBS/PBS (50 nM, 30 min, 37 • C), then visualized by fluorescence microscopy. Staining intensity was quantified in ImageJ and normalized to number of nuclei and osteoclast area.

Metabolites
Glucose and glutamine were measured using the Glucose (GO) Assay Kit and Glutamine/Glutamate Determination Kit. Lactate was assayed in heatinactivated medium by the increase in absorbance (340 nm) as NAD + was converted to NADH in the presence of 0.32 M glycine, 0.32 M hydrazine, 9.6 mM NAD + and 3 U/ml lactate dehydrogenase. The results were normalized to osteoclast number. Intracellular neutral lipid was detected in formalin-fixed cells washed with 60% isopropanol, air-dried, incubated in Oil Red O (70 mM) and washed and photographed in distilled water.

PDH activity
The PDH Enzyme Activity Microplate Assay (Abcam) was used to assess the ability of immunocaptured PDH to convert pyruvate to acetyl-CoA by following the reduction of NAD + to NADH [17].

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KJ Morten et al. 16 h post-transfection, the cells were exposed to experimental conditions. Luminesence was assayed using the Dual-Luciferase Reporter Assay System (Promega), with firefly luciferase normalized to the Renilla transfection control.

Statistics
Results are expressed as mean ± standard deviation (SD) of at least three independent experiments. Statistical analysis comprised one-way analysis of variance (ANOVA) using Bonferroni's multiple comparison as a post hoc test (except for experiments with only two conditions, for which a t-test was applied), with results considered significant at p < 0.05.

Hypoxia increases mitochondrial metabolic activity in osteoclasts
To investigate whether hypoxic osteoclasts produce additional energy for bone resorption, we measured intracellular ATP under normoxia and hypoxia (24 h, 2% O 2 ). When cultured on plastic, primary monocytes and osteoblasts, which share the osteoclast bone micro-environment, showed reduced intracellular ATP in line with published reports [6], whereas hypoxic osteoclasts increased intracellular ATP by 56% ( Figure 1A). When cultured instead on dentine, a substrate on which osteoclast resorption mechanisms are active, the hypoxic increase in intracellular ATP was not evident, suggesting that this ATP is utilized for bone resorption ( Figure 1A). We next assessed mitochondrial metabolic flux, assaying ETC activity using Alamar Blue [25]. Hypoxic osteoclasts rapidly increased ETC activity (125%, 4 h), reaching 169% at 24 h compared with decreased ETC activity in monocytes and osteoblasts ( Figure 1B). Unaltered mitochondrial porin expression ( Figure 1C) and nonyl-acridine orange staining (which binds mitochondrial cardiolipin; data not shown), suggested this was not due to increased mitochondrial mass. O 2 consumption remained considerable under hypoxia; indeed, ETC inhibition with rotenone had a greater effect under hypoxia (74% reduction) than in normoxic conditions (44% reduction; Figure 1D). In both environments O 2 consumption via the ETC. remained close to maximal, as compared with cells cultured in supplementary pyruvate ( Figure 1D). However, as probe sensitivity to changes in O 2 concentration is greater in the low O 2 range, we were unable to compare O 2 consumption rates at 20% and 2% O 2 directly.
HIF-1α siRNA reduced the hypoxic increase in ETC activity by 25% (Figure 2A), suggesting it to be partially HIF-1α-mediated. We therefore considered mitochondrial components of the HIF-mediated metabolic switch. COX subunit switching was evident, with increased COX4-2 (mRNA, protein) and reduced COX4-1 protein due to induction of the mitochondrial protease LONP1 ( Figure 2B, C). However, despite hypoxic induction of BNIP3 mRNA, BNIP3 protein was unchanged ( Figure 2B, C). This implies no stimulation of mitophagy, despite reduced expression of mitochondrial mRNAs, ATP synthase F0 subunit a/8 (ATP6/8 ) and cytochrome c oxidase subunit 3 (CO3 ) ( Figure 2B). (Glut-1 , SLC2A1 ) and glycolytic enzyme mRNA expression ( Figure 3A). Hypoxic transactivation of the PGK-1 hypoxia response element was HIF-1α-dependent, while HIF-2α exerted a small inhibitory effect ( Figure 3B). Hypoxia increased glucose uptake by osteoclasts (and primary monocytes and osteoblasts) in a HIF-1α-dependent manner ( Figure 3C, D). Hypoxia increased osteoclast lactate secretion, although the ratio of glucose consumption to lactate production was unaltered ( Figure 3C), indicative of increased flux through the glycolytic pathway but not of a switch to anaerobic glycolysis.

Active PDH in hypoxic osteoclasts
The PDH complex converts pyruvate to acetyl CoA and is permissive for continued mitochondrial metabolic flux. Phosphorylation by the HIF-1α target gene PDK1 inhibits PDH activity. Despite hypoxic induction of PDK1 mRNA (two-fold; p < 0.05), no consistent effect of hypoxia was observed on PDK1 protein expression in osteoclasts (data not shown). Hypoxia did not alter PDH activity ( Figure 4A) and neither did isoform-specific HIF siRNA affect PDH activity in either condition (data not shown). We therefore considered whether another regulator of cellular energy status, AMP-activated protein kinase (AMPK), might regulate PDH activity in osteoclasts. AMPK is activated by phosphorylation of Thr172 under metabolically stressful conditions, such as hypoxia [26]. However, AMPK phosphorylation was almost ablated in hypoxic osteoclasts ( Figure 4B). Chemical inhibition of AMPK with compound C similarly did not affect PDH activity ( Figure 4C, D). However, chemical activation of AMPK with metformin inhibited PDH, suggesting that osteoclast AMPK could inhibit PDH activity under hypoxia if it were activated. generation. Fatty acid oxidation has been proposed to drive ATP production in osteoclasts [23]. However, hypoxic osteoclasts accumulated intracellular neutral lipid ( Figure 5A), suggesting that hypoxia inhibits mitochondrial import and/or utilization of fatty acids [27,28]. Hypoxia did not affect lipid accumulation in monocytes or osteoblasts. Glutamine, a substrate for glutaminolysis, enters the mitochondrial TCA cycle at α-ketoglutarate. Hypoxic osteoclasts increased glutamine uptake 4.1-fold, whereas osteoblasts and monocytes inhibited and maintained uptake respectively ( Figure 5B). Hypoxic induction of glutamate uptake was HIF-2α-dependent ( Figure 5C). However, glutamine withdrawal had no effect on either ATP production or osteoclast survival, whereas removal of glucose dramatically inhibited both, especially in hypoxia ( Figure 5D).

Osteoclasts are sensitive to hypoxia-induced cell death
In contrast to other cells, osteoclasts are sensitive to even moderate hypoxia. 2% O 2 reduced osteoclast numbers to 83% and 65% of normoxic levels at 24 and 72 h respectively, whereas primary monocytes and osteoblasts continued to proliferate ( Figure 6A). We previously hypothesized the extent of osteoclast-mediated bone resorption under hypoxia to be a balance between osteoclast activation and osteoclast apoptosis [14]. Sensitivity to hypoxia-induced cell death might be a consequence of maintaining high rates of oxidative phosphorylation in a hypoxic environment, evidenced by increased expression of superoxide dismutase 2 (SOD2) ( Figure 6B), a marker of mitochondrial ROS formation. HIF-1α siRNA completely rescued osteoclasts from cell death induced by chronic (48 h) hypoxic exposure ( Figure 6C), whereas modulation of AMPK activity had no effect ( Figure 6D).

Discussion
We have demonstrated that hypoxic osteoclasts increase flux through both the glycolytic pathway and the mitochondrial ETC, driven by employment of selected components of the HIF-1α-mediated switch to anaerobic glycolysis. This enables osteoclasts to generate sufficient ATP to support enhanced bone resorption.
Glucose regulates osteoclast activity at normal physiological concentrations [21]. Increased glucose rapidly elevates the intracellular ATP:ADP ratio [22], while longer exposure activates vacuolar H + -ATPase (V-ATPase) A-subunit transcription to stimulate resorption [29]. This subunit directly interacts with the glycolytic enzyme phosphofructokinase-1 (PFK-1) [30]. Interaction between aldolase and the V-ATPase E-subunit also occurs in osteoclasts [31]. Both interactions are deemed mechanisms to micro-compartmentalize glycolytic ATP generation at the required intracellular location, directly linking glycolysis and osteoclast activation. Increased glycolysis by actively resorbing, hypoxic osteoclasts may also occur in vivo. Positron emission tomography (PET) with 2-(fluorine-18)fluoro-2-deoxy-D-glucose ( 18 FDG) can distinguish benign primary bone tumours containing many osteoclasts from those where osteoclasts are sparse [32], additionally correlating with markers of hypoxia [33]. Agents which inhibit glucose uptake/glycolysis inhibit bone resorption in animal models of disease [34,35] and can induce clinical remission in RA [36], suggesting targeted inhibition of glycolysis as a therapeutic anti-resorptive option. However, other cells within the hypoxic rheumatoid joint exhibit increased glycolytic capacity and the inhibitory activity of these agents does not solely target glycolysis, necessitating further investigation of the mechanism(s) behind such results. Glutamine consumption has not previously been described in osteoclasts. Hypoxia increased glutamine uptake, as in SK-N-SH neuroblastoma [37] and A549 lung adenocarcinoma cells [38], in a HIF-2αdependent manner. HIF-2α also drives the switch to hypoxic use of glutamine, rather than glucose, as the substrate for lipid synthesis [38]. Glutamine is additionally required for nucleotide and hexosamine biosynthesis. This complements our data that hypoxic osteoclasts require glucose for ATP production and cell survival but are insensitive to glutamine withdrawal, suggesting glucose as the primary substrate for ATP production, with glutamine potentially used for biosynthesis.
Osteoclasts only utilized selected components of the classical HIF-1α-mediated metabolic switch to anaerobic respiration. HIF-1α increased hypoxic glucose uptake and glycolytic flux and the HIF-1α-dependent COX subunit switch was observed. However, PDH was not inhibited and BNIP3 expression did not increase. These latter processes normally reduce hypoxic flux through the mitochondrial TCA cycle and ETC [10,11] and induce mitochondrial autophagy [12] respectively, both of which prevent toxic accumulation of ROS. As a result, mitochondrial ROS accumulates in hypoxic osteoclasts. However, ROS are essential for resorption. Tartrate-resistant acid phosphatase (TRAP) and cathepsin K co-localize in transcytotic vesicles. Digestion of TRAP by cathepsin K activates the ROSgenerating activity of TRAP to aid degradation of the products of bone digestion [39]. Osteoclast differentiation and resorption are enhanced by exogenous inducers of ROS; including H 2 O 2 [40], homocysteine [41] and hypoxia [42]. Indeed, this effect of hypoxia requires mitochondrial ROS and is reversed by the mitochondria-specific antioxidant MitoQ [42]. It is unclear why PDH was not inhibited in hypoxic osteoclasts. Despite HIF-1α induction, PDK1 mRNA was only modestly induced and we observed no consistent effect on PDK1 protein. PDH is also inhibited by hypoxic activation of AMPK and induction of PDK4 [43]. However, in osteoclasts hypoxia inhibited AMPK phosphorylation and so inactivated AMPK. AMPK is usually activated by reduced intracellular ratios of ATP:ADP or ATP:AMP, although hypoxic activation occurs independently of changes in intracellular energy status [26,44]. It may be that enhanced ATP production in hypoxic osteoclasts increases the intracellular ratio of ATP:AMP and overrides hypoxic mechanism(s) of AMPK activation in favour of dephosphorylation and inactivation. This would be necessary for hypoxic resorption to proceed, as AMPK inhibits osteoclast differentiation and activation [45]. Hypoxic attenuation of PDH activity is therefore prevented in osteoclasts by blockade of at least two pathways that usually contribute to its inhibition, allowing continued mitochondrial metabolic flux under hypoxia.
HIF-1α did not reduce mitochondrial metabolic flux, but did increase glycolytic flux and maintain the COX subunit switch, supporting our observation that increased hypoxic mitochondrial activity is partially HIF-1α-dependent. HIF-1α siRNA also prevented cell death during prolonged hypoxia. We hypothesize that in hypoxic osteoclasts, functional HIF-1α-dependent pathways initially increase ATP production and bone resorption [14]. However, lack of activation of HIF-1αdependent survival pathways eventually results in cell death. As osteoclasts are anyway short-lived cells, and as resorption cannot be allowed to continue indefinitely, allowing progressive accumulation of ROS in hypoxic osteoclasts may be an adaptive mechanism permitting rapid bone resorption in the short term, while ensuring curtailment of the process in the absence of re-oxygenation.
In summary, we have shown that hypoxia stimulates osteoclasts' consumption of glucose and glutamine and that increased glucose uptake is required for increased ATP production. HIF-1α-dependent stimulation of glucose uptake and glycolysis, in the absence of PDH inhibition by either HIF-1α or AMPK, drives high hypoxic mitochondrial ETC activity in these cells, although this eventually results in cell death. These mechanisms appear to support hypoxic induction of osteoclast resorption in the short term and might provide therapeutic targets for amelioration of the pathological bone resorption associated with diseases such as rheumatoid arthritis.