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Erythropoietin (EPO) is a pleiotropic cytokine that classically drives erythropoiesis but can also induce bone loss by decreasing bone formation and increasing resorption. Deletion of the EPO receptor (EPOR) on osteoblasts or B cells partially mitigates the skeletal effects of EPO, thereby implicating a contribution by EPOR on other cell lineages. This study was designed to define the role of monocyte EPOR in EPO-mediated bone loss, by using two mouse lines with conditional deletion of EPOR in the monocytic lineage. Low-dose EPO attenuated the reduction in bone volume (BV/TV) in Cx3cr1 Cre EPOR f/f female mice (27.05%) compared to controls (39.26%), but the difference was not statistically significant. To validate these findings, we increased the EPO dose in LysM Cre model mice, a model more commonly used to target preosteoclasts. There was a significant reduction in both the increase in the proportion of bone marrow preosteoclasts (CD115 + ) observed following high-dose EPO administration and the resulting bone loss in LysM Cre EPOR f/f female mice (44.46% reduction in BV/TV) as compared to controls (77.28%), without interference with the erythropoietic activity. Our data suggest that EPOR in the monocytic lineage is at least partially responsible for driving the effect of EPO on bone mass.

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 - Ly6C hi 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 (EPO) is a key regulator of erythropoiesis. However, EPO receptors (EPO-Rs) are also expressed on non-erythroid cell types, including myeloid and bone cells. Immune cells also participate in bone homeostasis. B cells produce receptor activator of nuclear factor kappa-Β ligand (RANKL) and osteoprotegerin (OPG), two pivotal regulators of bone metabolism. Here we explored the ability of B cells to transdifferentiate into functional osteoclasts and examined the role of EPO in this process in a murine model. Methods: We have combined specifically-designed experimental mouse models and in vitro based osteoclastogenesis assays, as well as PCR analysis of gene expression. Results: (i) EPO treatment in vivo increased RANKL expression in bone marrow (BM) B cells, suggesting a paracrine effect on osteoclastogenesis; (ii) B cell-derived osteoclastogenesis occured in vivo and in vitro, as demonstrated by B cell lineage tracing in murine models; (iii) B-cell-derived osteoclastogenesis in vitro was restricted to Pro-B cells expressing CD115/CSF1-R and is enhanced by EPO; (iv) EPO treatment increased the number of B-cell-derived preosteoclasts (β3 + CD115 + ), suggesting a physiological rationale for B cell derived osteoclastogenesis; (v) finally, mice with conditional EPO-R knockdown in the B cell lineage (cKD) displayed a higher cortical and trabecular bone mass. Moreover, cKD displayed attenuated EPO-driven trabecular bone loss, an effect that was observed despite the fact that cKD mice attained higher hemoglobin levels following EPO treatment. Conclusions: Our work highlights B cells as an important extra-erythropoietic target of EPO-EPO-R signaling and suggests their involvement in the regulation of bone homeostasis and possibly in EPO-stimulated erythropoietic response. Importantly, we present here for the first time, histological evidence for B cell-derived osteoclastogenesis in vivo ., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Recent studies have demonstrated that erythropoietin (EPO) treatment in mice results in trabecular bone loss. Here, we investigated the dose-response relationship between EPO, hemoglobin (Hgb) and bone loss and examined the reversibility of EPO-induced damage. Increasing doses of EPO over two weeks led to a dose-dependent increase in Hgb in young female mice, accompanied by a disproportionate decrease in trabecular bone mass measured by micro-CT (µCT). Namely, increasing EPO from 24 to 540 IU/week produced a modest 12% rise in Hgb (20.2 ± 1.3 mg/dL vs 22.7 ± 1.3 mg/dL), while trabecular bone volume fraction (BV/TV) in the distal femur decreased dramatically (27 ± 8.5% vs 53 ± 10.2% bone loss). To explore the long-term skeletal effects of EPO, we treated mice for two weeks (540 IU/week) and monitored bone mass changes after treatment cessation. Six weeks post-treatment, there was only a partial recovery of the trabecular microarchitecture in the femur and vertebra. EPO-induced bone loss is therefore dose-dependent and mostly irreversible at doses that offer only a minor advantage in the treatment of anemia. Because patients requiring EPO therapy are often prone to osteoporosis, our data advocate for using the lowest effective EPO dose for the shortest period of time to decrease thromboembolic complications and minimize the adverse skeletal outcome.

Erythropoietin (EPO) is the main hormone driving mammalian erythropoiesis, with activity mediated via the surface receptor, EPO-R, on erythroid progenitor cells. Recombinant human EPO is currently used clinically for the treatment of anemia in patients with end-stage renal disease, and in certain cancer patients suffering from anemia induced either by the tumor itself or by chemotherapy. EPO-R expression is also detected in non-erythroid cells, including macrophages present in the peritoneum, spleen, and bone marrow (BM). Here we demonstrate that Kupffer cells (KCs) - the liver-resident macrophages - are EPO targets. We show that, in vitro, EPO initiated intracellular signalling and enhanced phagocytosis in a rat KC line (RKC-2) and in sorted KCs. Moreover, continuous EPO administration in mice, resulted in an increased number of KCs, up-regulation of liver EPO-R expression and elevated production of the monocyte chemoattractant CCL2, with corresponding egress of Ly6C hi monocytes from the BM. In a model of acute acetaminophen-induced liver injury, EPO administration increased the recruitment of Ly6C hi monocytes and neutrophils to the liver. Taken together, our results reveal a new role for EPO in stimulating KC proliferation and phagocytosis, and in recruiting Ly6C hi monocytes in response to liver injury.

Erythropoietin (Epo) is the main hormone that regulates the production of red blood cells (hematopoiesis), by stimulating their progenitors. Beyond this vital function, several emerging roles have been noted for Epo in other tissues, including neurons, heart and retina. The skeletal system is also affected by Epo, however, its actions on bone are, as yet, controversial. Here, we review the seemingly contradicting evidence regarding Epo effects on bone remodeling. We also discuss the evidence pointing to a direct versus indirect effect of Epo on the osteoblastic and osteoclastic cell lineages. The current controversy may derive from a context-dependent mode of action of Epo, namely opposite skeletal actions during bone regeneration and steady-state bone remodeling. Differences in conclusions from the published in-vitro studies may thus relate to the different experimental conditions. Taken together, these studies indicate a complexity of Epo functions in bone cells., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Erythropoietin (Epo) is the main hormone that regulates the production of red blood cells (hematopoiesis), by stimulating their progenitors. Beyond this vital function, several emerging roles have been noted for Epo in other tissues, including neurons, heart, and retina. The skeletal system is also affected by Epo; however, its actions on bone are, as yet, controversial. Here, we review the seemingly contradicting evidence regarding Epo effects on bone remodeling. We also discuss the evidence pointing to a direct vs indirect effect of Epo on the osteoblastic and osteoclastic cell lineages. The current controversy may derive from a context-dependent mode of function of Epo, namely, opposite skeletal actions during bone regeneration and steady-state bone remodeling. Differences in conclusions deriving from the published in vitro studies may thus relate to the different experimental conditions. Taken together, the current state-of-the-art indicates definite Epo effects on bone cells and points to the complexity of the mode of function., (© 2017 Elsevier Inc. All rights reserved.)

The main oxygen sensor hypoxia inducible factor (HIF) prolyl hydroxylase 2 (PHD2) is a critical regulator of tissue homeostasis during erythropoiesis, hematopoietic stem cell maintenance, and wound healing. Recent studies point toward a role for the PHD2-erythropoietin (EPO) axis in the modulation of bone remodeling, even though the studies produced conflicting results. Here, we used a number of mouse strains deficient of PHD2 in different cell types to address the role of PHD2 and its downstream targets HIF-1α and HIF-2α in bone remodeling. Mice deficient for PHD2 in several cell lineages, including EPO-producing cells, osteoblasts, and hematopoietic cells (CD68:cre-PHD2 f/f ) displayed a severe reduction of bone density at the distal femur as well as the vertebral body due to impaired bone formation but not bone resorption. Importantly, using osteoblast-specific (Osx:cre-PHD2 f/f ) and osteoclast-specific PHD2 knock-out mice (Vav:cre- PHD2 f/f ), we show that this effect is independent of the loss of PHD2 in osteoblast and osteoclasts. Using different in vivo and in vitro approaches, we show here that this bone phenotype, including the suppression of bone formation, is directly linked to the stabilization of the α-subunit of HIF-2, and possibly to the subsequent moderate induction of serum EPO, which directly influenced the differentiation and mineralization of osteoblast progenitors resulting in lower bone density. Taken together, our data identify the PHD2:HIF-2α:EPO axis as a so far unknown regulator of osteohematology by controlling bone homeostasis. Further, these data suggest that patients treated with PHD inhibitors or EPO should be monitored with respect to their bone status. © 2016 American Society for Bone and Mineral Research., (© 2016 American Society for Bone and Mineral Research.)

Multiple myeloma (MM) is a plasma cell malignancy, characterized by osteolytic lesions and monoclonal immunoglobulins. The anemia, accompanying the disease is often treated with recombinant human EPO. Diverse non-erythropoietic effects of EPO have led us to question its combined action on the immune system and bone in the 5T33MM mouse model. EPO administration to MM mice attenuated disease progression as demonstrated by a decrease in serum MM IgG2b, splenic CD138 expressing cells, IL-6 and RORγτ transcripts in bone marrow (BM). IFN-γ transcript levels and macrophages (F4/80(+)CD11b(+)) in the BM both increased ~1.5 fold in the EPO-treated MM mice. In-vitro, EPO stimulated phagocytosis of 5T33MM cells (+30%) by BM-derived macrophages. In contrast, high-resolution microCT analysis of distal femurs revealed EPO-associated bone loss in both healthy and 5T33MM mice. EPO significantly increased expression of the osteoclastogenic nuclear factor-kappa B ligand (RANKL) in healthy mice, but not in MM mice, likely due to antagonizing effects on MM progression. Thus, in MM, EPO may act as a double-edged-sword stimulating immune response, while accelerating bone resorption, possibly via direct action on BM macrophages. This study supports a prudent approach of treating anemia in MM patients, aiming to maintain EPO-associated anti-MM effects, while considering bone damage.

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., (© FASEB.)