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

Abstract

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.

Figure 1

Hypoxia enhances mitochondrial metabolic activity. (A) Intracellular ATP assayed in primary human osteoclasts (OC), monocytes (MON) and osteoblasts (OB) following 24 h of culture in either normoxia (white bars) or hypoxia (2% O₂, grey bars); (right axis) relative amount of lacunar resorption following 24 h in hypoxia (black bar). Results are normalized to cell number and expressed relative to the normoxic level of ATP/resorption. (B) Relative Alamar Blue fluorescence following culture in normoxia (24 h, white bars) or hypoxia (2% O₂, 4 h or 24 h, grey bars). Results are normalized to cell number and expressed relative to the normoxic fluoresence. (C) Western blot of osteoclasts following 24 h of exposure to normoxia (N) or hypoxia (2% O₂, H). (D) Lifetime fluorescence values assessed as an indication of O₂ consumption rate in normoxic (white bars) and hypoxic (2% O₂, grey bars) conditions. Osteoclasts were cultured in either medium containing glucose (gluc) or medium lacking glucose but supplemented with 1 mm pyruvate (pyr), with or without rotenone (10 μM). *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 2

Effect of HIF on mitochondrial metabolic activity. (A) Relative Alamar Blue fluorescence following culture in normoxia (24 h, white bars) or hypoxia (2% O₂, 24 h, grey bars), following treatment with siRNA targeting HIF-1α (HIF-1), HIF-2α (HIF-2) or scrambled siRNA control (scr). Results are normalized to cell number and expressed relative to the hypoxic control fluoresence. (B) Hypoxic expression of mRNA versus β-actin mRNA (ACTB) control, shown relative to the normoxic level of expression. (C) Western blot of osteoclasts following 24 h of exposure to normoxia (N) or hypoxia (2% O₂, H). *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 3

Glucose uptake and glycolysis. (A) Hypoxic expression of mRNA versus β-actin mRNA (ACTB) control, shown relative to the normoxic level of expression. (B) PGK-1 HRE luciferase activation following 24 h of culture in normoxia (white bars) or hypoxia (2% O₂, 24 h, grey bars) following treatment with siRNA targeting HIF-1α (HIF-1), HIF-2α (HIF-2) or scrambled siRNA control (scr). Results are normalized to the Renilla transfection control and expressed relative to the hypoxic scrambled control. (C) Glucose consumption, lactate secretion and glucose consumption: lactate secretion ratio following 24 h of exposure to either normoxia (white bars) or hypoxia (2% O₂, grey bars). Results are normalized to cell number and expressed relative to the normoxic control. (D) Glucose consumption by osteoclasts following 24 h of culture in normoxia (white bars) or hypoxia (2% O₂, 24 h, grey bars) following treatment with siRNA targeting HIF-1α (HIF-1), HIF-2α (HIF-2) or scrambled siRNA control (scr). Results are normalized to cell number and expressed relative to the hypoxic control. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 4

PDH activity. (A) PDH activity following 24 h of exposure to either normoxia (white bar) or hypoxia (2% O₂, grey bar). Results are normalized to protein concentration and expressed relative to normoxic activity. (B) Western blot of osteoclasts following 24 h of exposure to normoxia (N) or hypoxia (2% O₂, H); n = 2. (C) Western blot following 24 h of exposure of osteoclasts to either compound C (10 μM, CmpC) or metformin (1 mm, Met). (D) PDH activity following 24 h of exposure to either normoxia (white bars) or hypoxia (2% O₂, grey bars) and either compound C (10 μM) or metformin (1 mm). Results are normalized to protein concentration and expressed relative to hypoxic activity. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5

Alternative substrates. (A) Quantified Oil Red O staining following 24 h of exposure to normoxia (N, white bars), hypoxia (2% O₂, H, light grey) or 100 μM oleic acid (dark grey bars). (Inset) Oil Red O staining of a representative osteoclast preparation (arrows indicate large, multinucleated osteoclasts). (B) Glutamine uptake following 24 h of exposure to either normoxia (white bars) or hypoxia (2% O₂, grey bars). Results are normalized to cell number and expressed relative to normoxic uptake. (C) Glutamine uptake following 24 h of culture in normoxia (white bars) or hypoxia (2% O₂, 24 h, grey bars) following treatment with siRNA targeting HIF-1α (HIF-1), HIF-2α (HIF-2) or scrambled siRNA control (scr). Results are normalized to cell number and expressed relative to the hypoxic control. (D) Intracellular ATP and osteoclast number assayed following 24 h of culture in either normoxia (white bars) or hypoxia (2% O₂, grey bars), with or without (w/o) either glucose or glutamine. ATP results are normalized to cell number and expressed relative to the hypoxic level of ATP. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 6

Osteoclast survival. (A) Relative number of osteoclasts, monocytes and osteoblasts following 24 h or 72 h of exposure to either normoxia (white bars) or hypoxia (2% O₂, grey bars). (B) Western blot of osteoclasts following 24 h of exposure to normoxia (N) or hypoxia (2% O₂, H). (C) Relative number of osteoclasts following 24 h or 48 h of exposure to either normoxia (white bars) or hypoxia (grey bars) following treatment with siRNA targeting HIF-1α (HIF-1), HIF-2α (HIF-2) or scrambled siRNA control (scr). (D) Treatment with compound C (10 μM) or metformin (1 mm). *p < 0.05; **p < 0.01; ***p < 0.001.