The Non-Erythropoietic EPO Analogue Cibinetide Inhibits Osteoclastogenesis In Vitro and Increases Bone Mineral Density in Mice

Abstract

The two erythropoietin (EPO) receptor forms mediate different cellular responses to erythropoietin. While hematopoiesis is mediated via the homodimeric EPO receptor (EPOR), tissue protection is conferred via a heteromer composed of EPOR and CD131. In the skeletal system, EPO stimulates osteoclast precursors and induces bone loss. However, the underlying molecular mechanisms are still elusive. Here, we evaluated the role of the heteromeric complex in bone metabolism in vivo and in vitro by using Cibinetide (CIB), a non-erythropoietic EPO analogue that exclusively binds the heteromeric receptor. CIB is administered either alone or in combination with EPO. One month of CIB treatment significantly increased the cortical (~5.8%) and trabecular (~5.2%) bone mineral density in C57BL/6J WT female mice. Similarly, administration of CIB for five consecutive days to female mice that concurrently received EPO on days one and four, reduced the number of osteoclast progenitors, defined by flow cytometry as Lin−CD11b−Ly6Chi CD115+, by 42.8% compared to treatment with EPO alone. In addition, CIB alone or in combination with EPO inhibited osteoclastogenesis in vitro. Our findings introduce CIB either as a stand-alone treatment, or in combination with EPO, as an appealing candidate for the treatment of the bone loss that accompanies EPO treatment.

Erythropoietin Receptor (EPOR) Signaling in the Osteoclast Lineage Contributes to EPO-Induced Bone Loss in Mice

“…Moreover, the deletion of EPOR in either osteoprogenitors/mature osteoblasts [ 21 , 22 ] or in B cells [ 40 ] attenuated EPO-driven bone loss but did not completely block the effect, suggesting that EPOR on other cell lineages also contributes to EPO-mediated bone loss. We previously demonstrated that high doses of EPO target the monocytic lineage by increasing the number of bone marrow preosteoclasts and bone resorption in vivo [ 18 , 26 , 42 ]. We also showed the direct stimulation of osteoclastogenesis by EPO in vitro [ 18 , 26 , 31 ], but the question of whether EPO-driven bone loss is mediated by EPOR activation on preosteoclasts in vivo remained elusive.…”

“…Our findings imply that EPOR in the monocytic lineage is responsible, at least in part, for driving the bone mass reduction caused by exogenously administered EPO. While these Cre lines do not discriminate between early precursors and late mature osteoclasts, we previously reported that while EPOR is highly expressed in osteoclast precursors, there is no expression or response to EPO and its analogs by mature osteoclasts [ 18 , 42 ]. It is therefore reasonable to assume that the skeletal response to EPO is partly driven by EPOR in osteoclast precursors.…”

“…Since recombinant erythropoietin is widely used in clinical practice to treat anemia associated with chronic kidney disease in patients who already suffer from compromised bone health, it is advisable to monitor these patients closely, and to minimize the potential adverse skeletal outcomes by administering the lowest effective dose of EPO for the shortest possible time [ 26 ]. Combining EPO with other anti-resorptive agents might also prove advantageous [ 42 ]. In addition, these findings should motivate the search for new alternatives to erythropoietin therapy, such as hypoxia-inducible factor-prolyl hydroxylase inhibitors.…”

“…However, the ablation of EPOR in these cell lineages did not completely abrogate the skeletal effect of EPO administration, suggesting the involvement of EPOR on additional cells. Recent results from our lab indicate that both high and physiologically relevant doses of EPO induce osteoclast differentiation in vitro by direct signaling through EPOR on osteoclast precursors [ 18 , 41 , 42 ]. This effect can be blocked by a specific non-erythropoietic EPO analog [ 42 ].…”

“…Recent results from our lab indicate that both high and physiologically relevant doses of EPO induce osteoclast differentiation in vitro by direct signaling through EPOR on osteoclast precursors [ 18 , 41 , 42 ]. This effect can be blocked by a specific non-erythropoietic EPO analog [ 42 ]. However, the question of whether EPOR on preosteoclasts directly contributes to EPO-driven bone loss in vivo has not yet been resolved.…”