Pyruvate dehydrogenase kinase 4 induces bone loss at unloading by promoting osteoclastogenesis☆
Highlights
► Pdk4−/− mice are resistant to bone loss at unloading. ► Pdk4 is upregulated in osteoblasts at unloading, regulates Rankl expression, and promotes osteoclastogenesis. ► Pdk4 in osteoclast precursors is also involved in osteoclast differentiation. ► Upregulation of Pdk4 in bone under unloaded conditions requires the osteocyte network. ► Pdk4 is, at least in part, responsible for disuse osteoporosis.
Introduction
Bone mass is determined by the balance between the activities of osteoblasts, which form bone, and those of osteoclasts, which resorb bone. Osteoporosis, which is one of the major age-related diseases in our modern world, is caused by the unbalance of these two activities, which are influenced by diet, physical activities, hormonal status, cytokines, and clinical status, such as diabetes mellitus and glucocorticoid treatment [1]. Disuse osteoporosis, which is caused by non-weight bearing, immobilization, or long-term bed rest, is rapidly increasing due to the increase in bedridden patients with age-associated diseases. Usually, the activities of osteoblasts and osteoclasts are coupled, and an increase in bone resorption enhances bone formation; however, in the case of disuse osteoporosis, the increased bone resorption does not enhance bone formation, indicating that the activities of osteoblasts were reduced but those of osteoclasts were enhanced in disuse osteoporosis [2]. Osteopontin, sympathetic nervous tone, sclerostin, and TRIPV4 have been shown to be associated with bone loss after unloading [3], [4], [5], [6].
Pyruvate dehydrogenase kinase isozymes (Pdk1, Pdk2, Pdk3, and Pdk4) are negative regulators of pyruvate dehydrogenase complex (PDC), which converts pyruvate to acetyl-CoA in the mitochondria, linking glycolysis to the energetic and anabolic functions of the tricarboxylic acid (TCA) cycle. PDC is a multi-subunit complex consisting of three catalytic domains: E1 (pyruvate decarboxylase), E2 (dihydrolipoamide acetyltransferase), and E3 (dihydrolipoamide dehydrogenase). Pdks phosphorylate the specific serine residues on E1 and inactivate PDC, whereas pyruvate dehydrogenase phosphatases (Pdp1 and Pdp2) dephosphorylate them and activate PDC. Short-term mechanisms of Pdk regulation include inhibition by the E1 substrate pyruvate and activation by the PDC products acetyl-CoA and NADH, although Pdk isoforms have different sensitivities against them. Mammalian Pdks exhibit tissue-specific expression: Pdk1 is expressed in the heart, pancreatic islets, and skeletal muscle; Pdk2 is ubiquitously expressed in the fed state with high expression in the heart, liver, and kidney; Pdk3 is mainly expressed in the testis, kidney, and brain; and Pdk4 is highly expressed in the heart, skeletal muscle, liver, kidney, and pancreatic islets [7], [8], [9]. The expression of Pdk4 in cardiac and skeletal muscles is rapidly upregulated during starvation and diabetes, which is reversed upon feeding and insulin administration, respectively [10], [11]. Pdk4 expression is also upregulated by glucocorticoid and is reversed by insulin [12], [13]. The active PDC is greater in the kidney, skeletal muscle, diaphragm, and heart but not in the liver of starved Pdk4−/− mice, and overexpression of Pdk4 in the heart exacerbates cardiomyopathy caused by the calcineurin stress-activated pathway [14], [15].
Bone adjusts its shape and strength against mechanical stress. Osteocytes are the most abundant cells in bone and comprise the communication system through the processes and canaliculi throughout bone. The osteocyte network is considered to be an ideal mechanosensor and mechanotransduction system [16], [17], [18], [19], [20]. We found that overexpression of BCL2 in osteoblasts reduces the number of osteocyte processes, probably due to the function of Bcl2 that modulates cytoskeletal reorganization [21], and induces the apoptosis of osteocytes, in which the transgene expression was reduced, presumably caused by an insufficient supply of oxygen, nutrients, and survival factors due to the reduced osteocyte processes (accompanying paper). The osteocyte apoptosis in BCL2 transgenic mice was gradually accumulated and the frequency of TUNEL-positive osteocyte lacunae reached to 75% at 4 months of age, and BCL2 transgenic mice at 4 months of age were resistant to bone loss after unloading (accompanying paper). By comparing wild-type and BCL2 transgenic mice, we searched for the genes responsible for bone loss after unloading, and found that Pdk4 expression is upregulated after unloading and down-regulated after reloading in wild-type mice but these regulations are never observed in BCL2 transgenic mice. We report here that upregulation of Pdk4 expression in osteoblasts and bone marrow cells after unloading is, at least in part, responsible for the enhancement of osteoclastogenesis and bone resorption after unloading.
Section snippets
Tail suspension and microarray
Tail suspension experiments were performed as previously described [22] with slight modification. Wild-type and BCL2 transgenic male mice at 10 weeks of age were placed in special cages for tail suspension (TS cages) and kept for one week to adapt to the TS cages. Tail suspension was performed for 7 days in the tail-suspended groups. Mice in the reloading groups were released from tail suspension for 24 h after tail suspension for one week. The control groups were under the same conditions except
Induction of Pdk4 expression in osteogenic cells after unloading
We searched the unloading-sensitive genes, whose expressions are upregulated after unloading and down-regulated after reloading, by tail suspension using wild-type mice and BCL2 transgenic mice, in which the osteocyte network was disrupted due to osteocyte apoptosis (accompanying paper). In microarray analysis using RNA from the tibiae and femurs, we found that Pdk4 was upregulated after unloading but down-regulated after reloading in wild-type mice, but Pdk4 was not upregulated after unloading
Discussion
The expression of Pdk4, whose protein regulates PDC activity and the supply of acetyl-CoA to the TCA cycle, was upregulated after unloading in osteogenic cells and bone marrow cells. Although the bone of Pdk4−/− mice normally developed and was maintained, it was resistant to bone loss after unloading. Bone resorption was enhanced with upregulated Rankl expression after unloading in wild-type mice but not in Pdk4−/− mice. Pdk4 positively regulated osteoclastogenesis through the induction of Rankl
Acknowledgments
We thank C. Fukuda for secretarial assistance. This work was supported by grants from the Japanese Ministry of Education, Culture, Sports, Science and Technology, the “Ground-based Research Program for Space Utilization” promoted by the Japan Space Forum, and the president's discretionary fund of Nagasaki University, Japan.
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All authors have no conflicts of interest.
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Present address: School of Basic Medical Science, Wuhan University, Wuhan, Hubei 430071, China.