Erythropoietin (EPO) is a key regulator of erythropoiesis, and it is mainly used to treat anemia. However, it is also administered prophylactically to non-anemic patients in certain clinical settings and is known to be used illicitly by athletes. The effect of EPO is controversial but emerging evidence indicates that EPO treatment induces bone loss in healthy mice. Here, we investigated the immediate and short-term skeletal effects of a single high-dose EPO injection in young mature (9 weeks) female mice. Cellular and molecular markers of bone turnover were evaluated at multiple time points post-injection. EPO administration led to a rapid increase in macrophage colony-stimulating factor (M-CSF) levels within the bone marrow (BM) microenvironment and in the serum, accompanied by an increase in BM CD115-positive cells and osteoclast precursors, as assessed by flow cytometry. This early cellular response to EPO was followed by an increase in tartrate-resistant acid phosphatase 5b (TRAP5b) and a decrease in procollagen type 1 N-terminal propeptide (P1NP), as determined by serum ELISA analyses, suggesting increased osteoclast numbers and decreased bone formation, respectively. Micro-computed tomography (μCT) revealed a significant reduction in trabecular bone volume. These findings demonstrate that even a single high-dose EPO injection disrupts bone homeostasis and induces significant bone loss through early modulation of the BM niche and osteoclastogenic pathways. Our results have important clinical implications for the prophylactic use of EPO and highlight potential skeletal risks.

Erythropoietin (EPO) is a principal regulator of erythropoiesis, however, its immediate effects on bone marrow (BM) are not fully resolved. The CXCR4–CXCL12 chemokine axis is critical for retaining hematopoietic cells within the BM niche, and its disruption is known to promote cell mobilization from the BM to the peripheral blood (PB). Here, we investigated the early BM response to EPO by treating C57BL/6 mice with a single 180 IU dose of EPO and analyzing the BM and PB 16 hours later. In the BM, EPO treatment resulted in a marked reduction of the general erythroid population (~32%), T cells (~57%), and B220high B cells (~25%), with a corresponding 47% increase of B220low B cells. Profiling of the BM erythroid subpopulations revealed a 42% decrease in reticulocytes (Ter119high, CD71-, FSC-Alow) accompanied by their 75% increase in the PB. Interestingly, ortho/polychromatic erythroblasts (Ter119high, CD71-, FSC-Ahigh) declined by 33% in BM without a corresponding increase in circulation, suggesting their rapid maturation induced by EPO. No significant change was observed in basophilic erythroblasts (Ter119high, CD71+, FSC-Ahigh). Transcriptional profiling of the CXCR4–CXCL12 axis revealed a 52% reduction in CXCR4 expression in BM hematopoietic cells and a 59% decrease in CXCL12 expression in BM stromal cells, implicating niche remodeling induced by the CXCR4-CXCL12 axis as a mediator of this rapid cellular response. These findings reveal that EPO induces an immediate remodeling of the BM microenvironment, driving the egress of erythroid and lymphoid populations through suppression of the CXCR4–CXCL12 BM retention axis. These data suggest that EPO functions not only as a proliferative hormone but also as an immediate effector of BM hematopoietic trafficking, with implications for both physiological stress erythropoiesis and clinical application of erythropoiesis-stimulating agents (ESA's).

Colon carcinoma is among the most prevalent malignant tumors, with inflammation being the primary risk factor. Cannabinoid receptor 2 (CB2/CNR2) has complex immunomodulatory functions. Therefore, we investigated the role of osteogenic growth peptide (OGP), an endogenous selective CB2 agonist, in colon carcinogenesis and immune modulation in transgenic mice (ApcMin/+).We injected 8-week-old (progression phase) or five-week-old (initiation phase) ApcMin/+ mice with OGP or vehicle weekly for 8 weeks or 4 weeks, respectively. During the progression phase, OGP-treated mice displayed significantly fewer tumors in the large intestine and smaller tumors in the small intestine. During the initiation phase, OGP significantly attenuated adenomagenesis in both the small and large intestine, decreased IL-6 and IL-4 levels, increased splenic anti-tumor CD8+ T cells, and diminished populations of tumor-promoting myeloid-derived suppressor cells. Further, we used exomic analyses of UKBiobank patients to determine the relationship between CNR2 polymorphisms and tumor-associated myeloid cells in humans. We found that the common CNR2-Q63R polymorphism is associated with monocyte count. Our results suggest that CB2 activation via OGP attenuates tumorigenesis and adenoma growth by modulating immune cells, corroborated by a significant association between CNR2 polymorphisms and monocytopoiesis in humans.

Approximately one third of all newly synthesized proteins are estimated to be processed through the secretory pathway. This complex process presents multiple opportunities for regulation of protein production and function. Current examples of the differential regulation of translocation of specific polypeptides across the Endoplasmic Reticulum (ER) membrane, have focused on the responses to ER stress. Differences in the folding surrounding between the cytosol and the ER lumen, prevent mislocalized proteins from properly folding thus making them highly toxic to the cell. As such, mislocalized proteins are subjected to proteasomal degradation by the pre-emptive quality control (pQC) process which is viewed as part of the unfolded protein response (UPR). Accumulatively, the various UPR cellular process aim to maintain ER homeostasis during changes in physiological or stress conditions. Here we used a specific ER translocation inhibitor, CAM741 (Novartis), to demonstrate that the regulated translocation of the erythropoietin receptor (EPOR) into the ER lumen responds to erythropoietin hormone levels. Our results suggest a new mode of regulation by which extra-cellular signaling can affect the entry of specific nascent chains into the ER lumen. Uncovering the mechanism by which extra-cellular conditions regulate ER translocation of a specific polypeptide has potential as a means of intervention, with potential clinical implications.

Bone tissue constantly undergoes a process of remodeling that simultaneously involves bone resorption by osteoclasts and bone formation by osteoblasts. Osteoclasts are multinuclear cells that originate primarily from myeloid precursors, while macrophage colony-stimulating factor (M-CSF) is essential for their survival and differentiation. Osteoblasts originate from mesenchymal cells and differentiate into osteocytes (mineralized bone resident cells). The balance between bone resorption and bone formation determines changes in bone mass. A wide variety of therapeutic interventions can affect bone metabolism. Erythropoietin (EPO), a primary regulator of red cell lineage, is commonly used in the clinic to treat anemia in certain clinical conditions and is sometimes administered to non-anemic patients before surgery to reduce the need for perioperative blood transfusion. We have previously shown that multidose (2-week) exposure to EPO significantly reduces bone mass in mice. In this study, we explored the dynamics of the response to a single high-dose EPO treatment in bone and bone marrow (BM) microenvironment in mice. Bone parameters were examined at four time points (16 hours, 48 hours, 1 week, and 2 weeks) following a single EPO injection (180 U) in young (9 weeks) female mice. We found that this single dose of EPO led to a 32% decrease in bone volume/total volume (BV/TV) in the distal femur within 1 week, as assessed by µCT analysis. Serum M-CSF levels increased by 180% 16 hours after EPO administration. This increase was accompanied by a 2-fold increase in soluble M-CSF in the BM extracellular matrix, which returned to baseline levels 48 hours after EPO administration. M-CSF-receptor (CD115) positive cells in the BM increased by 32% and 39% 48 hours and 1 week following EPO injection, respectively. Osteoclast precursor cells (Lin− CD11b− Ly6Chi CD115+) increased by 32%, 32% and 46%, two days, 1 week and 2 weeks after EPO injection, respectively. In line with these findings, serum levels of tartrate resistance acid phosphatase 5b (TRAP5b), a marker of bone-resorbing osteoclasts, increased in EPO-treated mice by ~20% 1 week after EPO injection, compared to diluent injected controls. Bone formation was also significantly affected by EPO treatment, as reflected by a ~80% decrease in procollagen type I N-terminal propeptide (P1NP) within 48 hours after EPO injection and returned to baseline one-week post-treatment. This study sheds light on the dynamic bone response to EPO, showing for the first time that even a single dose of EPO may have significant implications for bone metabolism. These findings are highly relevant in the case of EPO administration to non-anemic patients before surgery for potential avoidance of allogeneic transfusions.

Erythrocyte biogenesis needs to be tightly regulated to secure oxygen transport and control plasma viscosity. The cytokine erythropoietin (Epo) governs erythropoiesis by promoting cell proliferation, differentiation, and survival of erythroid precursor cells. Erythroid differentiation is associated with an accumulation of the cyclin–dependent kinase inhibitor p27Kip1, but the regulation and role of p27 during erythroid proliferation remain largely unknown. We observed that p27 can bind to the erythropoietin receptor (EpoR). Activation of EpoR leads to immediate Jak2–dependent p27 phosphorylation of tyrosine residue 88 (Y88). This modification is known to impair its CDK–inhibitory activity and convert the inhibitor into an activator and assembly factor of CDK4,6. To investigate the physiological role of p27–Y88 phosphorylation in erythropoiesis, we analyzed p27Y88F/Y88F knock–in mice, where tyrosine–88 was mutated to phenylalanine. We observed lower red blood cell counts, lower hematocrit levels, and a reduced capacity for colony outgrowth of CFU–Es (colony–forming unit–erythroid), indicating impaired cell proliferation of early erythroid progenitors. Compensatory mechanisms of reduced p27 and increased Epo expression protect from stronger dysregulation of erythropoiesis. These observations suggest that p27–Y88 phosphorylation by EpoR pathway activation plays an important role in the stimulation of erythroid progenitor proliferation during the early stages of erythropoiesis.

The impact of erythropoietin (EPO) on erythropoiesis has long been established. However, recent studies have indicated that EPO may also exert regulatory effects on other hematopoietic lineages, including the monocyte lineage. In that respect, we have previously shown that EPO treatment in mice induces bone loss. Here, we studied the effect of EPO on bone marrow (BM) monocytes and its potential implications for osteoclastogenesis. To address the dynamic changes in monocyte populations following EPO administration, we injected EPO (180U) once, or 3 times on alternating days for one week. Mice were sacrificed 16 h after EPO injection. Our results revealed a significant 20 percent increase in the monocyte population (TER119 -, LY6G -, CD11b +) 16 hours after EPO injection, and a twofold increase in the levels of these cells after one week of 3 injections of EPO. Soluble macrophage colony-stimulating factor (MCSF) protein levels in the bone marrow were increased 1.5-fold 16 h after EPO injection, while MCSF mRNA expression levels remained unchanged. After 1-week of EPO administration (3 injections), although the global BM expression of MCSF was reduced, we observed a two-fold increase in MCSF expression levels in T cells (CD3 +). To further elucidate the functional consequences of EPO-induced monocyte expansion, we isolated monocytes from mice treated with EPO for one week. We characterized expression levels of key osteoclastic markers in these cells. Indeed, we found that in BM monocytes, EPO treatment led to the upregulation of Lymphocyte function-associated antigen 1 (LFA-1) and Receptor Activator of Nuclear Factor κ B (RANK). The former is implicated in osteoclastogenesis via its interaction with intercellular adhesion molecule-1 (ICAM−1), while the latter is the surface receptor for RANK ligand which regulates osteoclast differentiation and activation. These data suggest that EPO not only influences BM monocyte expansion, but also contributes to the regulation of osteoclast differentiation, thus linking erythropoiesis to bone homeostasis. Furthermore, we investigated the early effects of EPO treatment on the expression of transcription factors implicated in monocyte differentiation. Remarkably, we found that EPO administration for 16 hours resulted in the downregulation of KLF2 and KLF4, transcription factors involved in monocytic lineage commitment. The rapid EPO-induced expansion of the monocytic population, coupled with the EPO-regulated expression of MSCF, osteoclastic markers, and transcription factors, suggests a role for EPO in modulating monocyte-to-osteoclast differentiation. The current study opens avenues for further investigating the crosstalk between erythropoiesis, myelopoiesis, and bone homeostasis.

A series of 1,8-Naphthyridines were synthesized from the starting material, 2-Aminonicotinaldehyde (also called as 2-amino- Pyridine-3- carbox aldehyde) through Friedlander annulation. The reaction proceeds in presence of eco-friendly reusable Poly Ethyelene Glycol-400(PEG:400) solvent in catalyst free conditions, shorter reaction times and in good yields. All the synthesized compounds were characterized by IR, 1H NMR and Mass spectral analyses.

Five million non-melanoma skin cancers occur globally each year, and it is one of the most common malignant cancers. The dysregulation of the endocannabinoid system, particularly cannabinoid receptor 2 (CB2), is implicated in skin cancer development, progression, and metastasis. Comparing wildtype (WT) to systemic CB2 knockout (CB2-/-) mice, we performed a spontaneous cancer study in one-year old mice, and subsequently used the multi-stage chemical carcinogenesis model, wherein cancer is initiated by 7,12-dimethylbenz[a]anthracene (DMBA) and promoted by 12-O-tetradecanoylphorbol-13-acetate (TPA). We found that aging CB2-/- mice have an increased incidence of spontaneous cancerous and precancerous skin lesions compared to their WT counterparts. In the DMBA/TPA model, CB2-/- developed more and larger papillomas, had decreased spontaneous regression of papillomas, and displayed an altered systemic immune profile, including upregulated CD4+ T cells and dendritic cells, compared to WT mice. Immune cell infiltration in the tumor microenvironment was generally low for both genotypes, although a trend of higher myeloid-derived suppressor cells was observed in the CB2-/- mice. CB2 expression in carcinogen-exposed skin was significantly higher compared to naïve skin in WT mice, suggesting a role of CB2 on keratinocytes. Taken together, our data show that endogenous CB2 activation plays an anti-tumorigenic role in non-melanoma skin carcinogenesis, potentially via an immune-mediated response involving the alteration of T cells and myeloid cells coupled with the modulation of keratinocyte activity.

Background: Erythropoietin (EPO) is a glycoprotein hormone which serves as a key regulator of erythropoiesis. Previous studies have shown that it also has a myriad of extraerythropoietic effects including on immune and skeletal systems. Specifically, it was shown that EPO reduces bone marrow (BM) B cell population, in part, by inducing an egress of IgM+ cells out of BM to the peripheral blood (PB). However, the effect of EPO on early B cells (IgM- Pro- and Pre-B cells) has not been studied. Moreover, a process of transdifferentiation of early B cells into erythroid cells under EPO stimulation has not been addressed. Methods: We used MB1-Cre;R26-eYFP mice for B-cell-specific lineage tracing as well as MB1-Cre; EPO-Rfl/fl and Osx-Cre; EPO-Rfl/fl mouse models for B-cell and osteoblast-specific EPO-R knockdown, respectively. Mice (10-12 week old females) were treated with either diluent or human recombinant EPO (hereafter, EPO) for one week (180IU x3/week). Immunophenotyping of BM cells was performed by multi-color flow cytometry (mFC) and immunofluorescence microscopy (IF) was used to demonstrate B cell-derived erythropoiesis in BM sections. Expression of PAX5 and IL-7 in the BM was determined by real-time quantitative PCR. Results: EPO treatment led to a significant 2.4-fold reduction in BM B cells (B220+/CD19+) manifested by a decrease in both IgM+ and IgM- cell fractions. While the IgM+ B cell fraction in PB after one week of EPO did not change, we did observe an almost 2-fold increase in IgM- B cells in EPO-treated as compared to diluent-treated mice (1.4±0.8% vs 0.7±0.5%, respectively, p<0.05). There was no change in IgM+ or IgM- B cells in the spleen, suggesting that IgM- B cells in PB originate from the BM. Concurring with these data, expression of CXCR4 adhesion molecule by BM Pro-B (IgM-) cells was significantly reduced (1.8-fold) upon EPO treatment. Since a decrease in BM B cells could not be solely explained by their egress from BM, we hypothesized that EPO induced a maturation delay at the level of Pre/Pro-B cell stages. Using both MB1-Cre; EPO-Rfl/fl and Osx-Cre; EPO-Rfl/fl mouse models we showed that this maturation delay is independent of EPO-EPO-R signaling in both early B cells and osteoblasts, i.e. following EPO treatment the amount and subpopulation distribution of BM B cells were similar in the control and corresponding EPO-R knockdown mice. However, we found significant perturbations in the key regulators of B cell lymphopoiesis in EPO-treated mice (EPO-R+/+): a 4-fold and 1.7-fold decrease in the expression of PAX5 and IL-7, respectively, while expression of IL-7 receptor by BM cells (both B220+ and B220-)was modestly but significantly reduced (as measured by mFC). Finally, applying IF on BM sections, we could detect eYFP+ BM cells (derived from MB1-Cre;R26-eYFP mice) that co-expressed the specific erythroid marker TER119 (Figure 1). These findings demonstrate that a fraction of BM B cells can transdifferentiate into erythroid cells upon EPO stimulation. Conclusions: Besides its effect on erythropoiesis, EPO compromises BM B cell compartment by a multifaceted mechanism, including BM egress, delay in maturation as well as transdifferentiation to the erythroid lineage. An understanding of the biological and clinical significance of this phenomenon requires further research. For example, the transdifferentiation might explain an improved erythropoiesis in patients with anemia who already have high EPO levels. This research was funded by Israel Science Foundation (ISF) grant number 1188/21 to DN. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Erythropoietin receptor (EPOR) is widely expressed in healthy and malignant tissues. In certain malignancies, EPOR stimulates tumor growth. In healthy tissues, EPOR controls processes other than erythropoiesis, including mitochondrial metabolism. We hypothesized that EPOR also controls the mitochondrial metabolism in cancer cells. To test this hypothesis, we generated EPOR-knockdown cancer cells to grow tumor xenografts in mice and analyzed tumor cellular respiration via high-resolution respirometry. Furthermore, we analyzed cellular respiratory control, mitochondrial content, and regulators of mitochondrial biogenesis in vivo and in vitro in different cancer cell lines. Our results show that EPOR controls tumor growth and mitochondrial biogenesis in tumors by controlling the levels of both, pAKT and inducible NO synthase (iNOS). Furthermore, we observed that the expression of EPOR is associated with the expression of the mitochondrial marker VDAC1 in tissue arrays of lung cancer patients, suggesting that EPOR indeed helps to regulate mitochondrial biogenesis in tumors of cancer patients. Thus, our data imply that EPOR not only stimulates tumor growth but also regulates tumor metabolism and is a target for direct intervention against progression.

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 Cx3cr1Cre EPORf/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 LysMCre 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 LysMCre EPORf/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.

EDITORIAL article Front. Cell Dev. Biol., 22 October 2021Sec.Cellular Biochemistry https://doi.org/10.3389/fcell.2021.769320

Background and aims: Erythropoietin (EPO) is a pleiotropic cytokine, which besides its classical role in driving erythropoiesis, displays tissue protective and immunomodulatory activities. EPO also induces bone loss. While hematopoiesis is mediated via the homodimeric EPO receptor (EPOR), tissue protection is conferred via a heteromer composed of EPOR and CD131. Cibinetide (CIB), a non-erythropoietic analogue of EPO, specifically binds to the heteromeric receptor and confers tissue protection. Our published findings that EPO stimulates osteoclast precursors and entrains a decrease in bone density, raise questions regarding the underlying molecular mechanisms. Here, we evaluated the role of the heteromeric complex in bone metabolism using CIB alone and in combination with EPO in vivo and in vitro.

In vitro osteoclastogenesis is a central assay in bone biology to study the effect of genetic and pharmacologic cues on the differentiation of bone resorbing osteoclasts. To date, identification of TRAP+ multinucleated cells and measurements of osteoclast number and surface rely on a manual tracing requiring specially trained lab personnel. This task is tedious, time-consuming, and prone to operator bias. Here, we propose to replace this laborious manual task with a completely automatic process using algorithms developed for computer vision. To this end, we manually annotated full cultures by contouring each cell, and trained a machine learning algorithm to detect and classify cells into preosteoclast (TRAP+ cells with 1–2 nuclei), osteoclast type I (cells with more than 3 nuclei and less than 15 nuclei), and osteoclast type II (cells with more than 15 nuclei). The training usually requires thousands of annotated samples and we developed an approach to minimize this requirement. Our novel strategy was to train the algorithm by working at “patch-level” instead of on the full culture, thus amplifying by >20-fold the number of patches to train on. To assess the accuracy of our algorithm, we asked whether our model measures osteoclast number and area at least as well as any two trained human annotators. The results indicated that for osteoclast type I cells, our new model achieves a Pearson correlation (r) of 0.916 to 0.951 with human annotators in the estimation of osteoclast number, and 0.773 to 0.879 for estimating the osteoclast area. Because the correlation between 3 different trained annotators ranged between 0.948 and 0.958 for the cell count and between 0.915 and 0.936 for the area, we can conclude that our trained model is in good agreement with trained lab personnel, with a correlation that is similar to inter-annotator correlation. Automation of osteoclast culture quantification is a useful labor-saving and unbiased technique, and we suggest that a similar machine-learning approach may prove beneficial for other morphometrical analyses.

Background: Murine models of myelodysplastic syndromes (MDS) exhibit lower bone mass (B-Mass), and several reports suggest increased incidence of osteoporosis and fractures in MDS patients. Whether MDS is associated with lower B-Mass is unknown.

High erythropoietin (Epo) levels are detrimental to bone health in adult organisms. Adult mice receiving high doses of Epo lose bone mass due to suppressed bone formation and increased bone resorption. In humans, high serum Epo levels are linked to fractures in elderly men. Our earlier studies indicated that Epo modulates osteoblast activity; however, direct evidence that Epo acts via its receptor (EpoR) on osteoblasts in vivo is still missing. Here, we created mice lacking EpoR in osteoprogenitor cells to specifically address this gap. Deletion of EpoR in osteoprogenitors ( EpoR:Osx-cre , cKO) starting at 5 weeks of age did not alter red blood cell parameters but increased vertebral bone volume by 25% in 12-week-old female mice. This was associated with low bone turnover. Histological (osteoblast number, bone formation rate) and serum (P1NP, osteocalcin) bone formation parameters were all reduced, as were the number of osteoclasts and TRAP serum level. Differentiation of osteoblast precursors isolated from cKO versus control mice resulted in lower expression of osteoblast marker genes including Runx2, Alp, and Col1a1 on day 21, whereas the mineralization capacity was similar. Moreover, the RANKL/OPG ratio, which determines the osteoclast-supporting potential of osteoblasts, was substantially decreased by 50%. Similarly, coculturing cKO osteoblasts with control or cKO osteoclast precursors produced significantly fewer osteoclasts than coculture with control osteoblasts. Finally, exposing female mice to Epo pumps (10 U·d −1 ) for 4 weeks resulted in trabecular bone loss (−25%) and increased osteoclast numbers (1.7-fold) in control mice only, not in cKO mice. Our data show that EpoR in osteoprogenitors is essential in regulating osteoblast function and osteoblast-mediated osteoclastogenesis via the RANKL/OPG axis. Thus, osteogenic Epo/EpoR signaling controls bone mass maintenance and contributes to Epo-induced bone loss.

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.