Erythropoietin directly stimulates osteoclast precursors and induces bone loss

Abstract

Erythropoietin (EPO) primarily regulates red blood cell formation, and EPO serum levels are increased on hypoxic stress (e.g., anemia and altitude). In addition to anemia, recent discoveries suggest new therapeutic indications for EPO, unrelated to erythropoiesis. We investigated the skeletal role of EPO using several models of overexpression (Tg6 mice) and EPO administration (intermittent/continuous, high/low doses) in adult C57Bl6 female mice. Using microcomputed tomography, histology, and serum markers, we found that EPO induced a 32%-61% trabecular bone loss caused by increased bone resorption (+60%-88% osteoclast number) and reduced bone formation rate (-19 to -74%; P < 0.05 throughout). EPO targeted the monocytic lineage by increasing the number of bone monocytes/macrophages, preosteoclasts, and mature osteoclasts. In contrast to the attenuated bone formation in vivo, EPO treatment in vitro did not inhibit osteoblast differentiation and activity, suggesting an indirect effect of EPO on osteoblasts. However, EPO had a direct effect on preosteoclasts by stimulating osteoclastogenesis in isolated cultures (+60%) via the Jak2 and PI3K pathways. In summary, our findings demonstrate that EPO negatively regulates bone mass and thus bears significant clinical implications for the potential management of patients with endogenously or therapeutically elevated EPO levels.

Polycythemia is associated with bone loss and reduced osteoblast activity in mice

“…Impaired osteogenesis was recently described in the tg6 mouse by other investigators and was likewise reported in animal models of thalassemia [31, 40–42]. In this disorder, a defective hemoglobin molecule leads to continuous erythroid destruction and anemia, which then causes a rise in circulating EPO and a subsequent expansion of defective erythroids, in a vicious circle of “ineffective erythropoiesis”.…”

“…However the results on the skeleton are controversial. One group of investigators showed a positive effect on bone mass and bone formation in newborn and older animals treated with EPO [12] while two other groups observed that exogenous EPO administration resulted in bone loss caused by increased bone turnover [30, 31]. The debate extends on the potential mechanisms of EPO action in vivo and whether EPO has direct humoral effects on bone homeostasis.…”

“…This transgenic animal constitutively overexpresses EPO in an oxygen-independent manner [34, 35]. This mouse has a 10–12-fold elevation in EPO plasma levels [34] resulting in erythrocytosis [36] along with a decrease in bone mass [31]. Herein, we compared the bone phenotype of these two mice to determine the potential effects of EPO on the skeleton beyond polycythemia.…”

Scaling of titanium implants entrains inflammation-induced osteolysis

“…Primary bone marrow-derived macrophages (BMDMs) were isolated from the femora and tibiae of adult C57BL/6J-RCC mice, as previously described21. Briefly, cells were cultured overnight in 6-well dishes at 37 °C in a humidified atmosphere with 5% CO 2 in alpha modified Eagle’s medium (αMEM, Life Science Technology, NY, USA).…”

“…For the osteoclastogenesis assay, BMDM, which are the same as preosteoclasts in vitro , were plated in 96-well plates (7,000 cells per well) in standard medium supplemented with 20 ng/ml M-CSF and 50 ng/ml RANKL (R&D Systems, Minneapolis, MN, USA), replaced every 2 days, as previously described21. Treatments (either LPS or titanium particles) were added after two days of incubation.…”