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Oxidative stress is involved in the deterioration of bone quality and mechanical strength in both diabetic and aging adults. Therefore, we studied the ability of the antioxidant compound, S-allylmercapto- N-acetylcysteine (ASSNAC) to protect bone marrow stromal cells (BMSCs) from advanced glycation end-products (AGEs) cytotoxicity and improve bone microarchitecture of adult healthy and obese/diabetic (db/db) female mice. ASSNAC effect on AGEs-treated cultured rat BMSCs was evaluated by Neutral Red and XTT cell survival and reactive oxygen species (ROS) level assays. Its effect on healthy (C57BL/6) and obese/diabetic (C57BLKS/J Leprdb+/+; db/db) female mice femur parameters, such as (1) number of adherent BMSCs, (2) percentage of CD73+/CD45− cells in bone marrow (BM), (3) glutathione level in BM cells, and (4) femur microarchitecture parameters by microcomputed tomography, was studied. ASSNAC treatment protected BMSCs by significantly decreasing AGEs-induced ROS production and increasing their cellular resistance to the cytotoxic effect of AGEs. ASSNAC treatment of healthy female mice (50 mg/kg/day; i.p.; age 12–20 weeks) significantly increased the number of BMSCs (+60%), CD73+/CD45− cells (+134%), and glutathione level (+110%) in the femur bone marrow. Furthermore, it increased the femur length (+3%), cortical diameter (+3%), and cortical areal moment of inertia (Ct.MOI; +10%) a surrogate for biomechanical strength. In db/db mice that demonstrated a compromised trabecular bone and growth plate microarchitecture, ASSNAC treatment restored the trabecular number (Tb.N, +29%), bone volume fraction (Tb.BV/TV, +130%), and growth plate primary spongiosa volumetric bone mineral density (PS-vBMD, +7%) and thickness (PS-Th, +18%). In conclusion, this study demonstrates that ASSNAC protects bone marrow cells from oxidative stress and may improve bone microarchitecture in adult healthy and diabetic female mice.

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 (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.

Krox20/EGR2 is a zinc finger transcription factor, implicated in the development of the hindbrain, nerve myelination, and tumor suppression. In skeletal biology, we have demonstrated that Krox20 also regulates adult bone metabolism. We and others have characterized several functions of Krox20 in the osteoclast lineage, namely, preosteoclast proliferation and differentiation, and mature osteoclast apoptosis. We have previously reported that systemically Krox20-haploinsufficient mice have a low bone mass with increased bone resorption. However, new data have now revealed that this phenotype is restricted to females. In addition, we discovered that conditional knockout of Krox20 (cKO) restricted to osteoclast progenitors is sufficient to induce the same female-specific bone loss observed in systemic mutants. To test whether this sexual dimorphism results from an interaction between Krox20 and sex hormones, we examined the sex- and hormone-dependent role of Krox20 deficiency on proliferation and apoptosis in osteoclastic cells. Our results indicate that male and female sex hormones (dihydrotestosterone [DHT] and estradiol [E2], respectively) as well as Krox20 inhibit preosteoclast proliferation and augment osteoclast apoptosis. The observation that Krox20 expression is inhibited by DHT and E2 negates the hypothesis that the effect of sex hormones is mediated by an increase in Krox20 expression. Interestingly, the effect of Krox20 deficiency was observed only with cells derived from female animals, regardless of any sex hormones added in vitro. In addition, we have identified sexual dimorphism in the expression of several Krox20-related genes, including NAB2. This sex-specific epigenetic profile was established at puberty, maintained in the absence of sex hormones, and explains the female-specific skeletal importance of Krox20. The findings described in this study emphasize the medical importance of sex differences, which may be determined at the epigenetic level. © 2019 American Society for Bone and Mineral Research.

Background Spontaneous catch-up (CU) growth occurs when a growth-restricting factor is resolved. However, its efficiency is sometimes inadequate and growth deficits remain permanent. The therapeutic toolbox for short stature is currently very limited, thus, finding new regulatory pathways is important for the development of novel means of treatment. Our previous studies using a nutrition-induced CU growth model showed that the level of sirtuin-1 (Sirt1) was significantly increased in food-restricted animals and decreased during CU growth. Aim This study sought to investigate the role of Sirt1 in modulating the response of the epiphyseal growth plate (EGP) to nutritional manipulation. Method Collagen type II-specific Sirt1 knockout (CKO) mice were tested for response to our CU growth model consisting of a period of food restriction followed by re-feeding. Results The transgenic CKO mice weighed more than the control (CTL) mice, their EGP was higher and less organized, specifically at the resting and proliferative zones, leading to shorter bones. Ablation of Sirt1 in the chondrocytes was found to have a dramatic effect on bone mineralization on micro-CT analysis. The CKO mice were less responsive to the nutritional manipulation, and their CU growth was less efficient. They remained shorter than the CTL mice who corrected the food restriction-induced growth deficit during the re-feeding period. Conclusions Sirt1 appears to be important for normal regulation of the EGP. In its absence, the EGP is less organized and CU growth is less efficient. These results suggest that SIRT1 may serve as a novel therapeutic target for short stature.

The long term skeletal effects of antenatal exposure to teratogen 5-deoxy-2′-cytidine (5-AZA) were studied using two inbred strains, C3H/HeJ (C3H, with inherently stronger bones) and C57Bl/6J (C57, with weaker bones). We previously reported that in-utero exposure to 5-AZA resulted in loss of bone quality in 3- and 6-mo-old C3H offspring. In this study, we further examined whether the long-term effects of an acute teratogenic exposure are still evident in older mice. Bone phenotypes of 12 mo-old mice exposed to a single injection of 5-AZA on day 10 of their mother's pregnancy were evaluated by micro-computed tomography and compared to the untreated controls.The main observation of this study is that 5-AZA-induced loss of bone length was registered in 12-mo-old C57 and C3H males. As expected, we did not find differences in the 3rd lumbar vertebra since in-utero exposure to 5-AZA was shown to affect the limb buds but not the axial skeleton. Trajectory of changes in bone phenotypes from ages 3 mo through 6 mo to 12 mo was also compared; 5-AZA-exposed C57 males had consistently lower femoral length and trabecular BMD than age-matched controls. In summary, by characterizing teratogen-exposed C57 and C3H mice, we further confirmed that the adaptive response to antenatal insults continue into mid-life of the mice as well as there is a sex-specificity of these responses. Keywords: Bone loss, Genetic heterogeneity, Adult mice, Developmental origin of diseases

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. Results: CIB injections to 12-week-old female mice (120 µg/kg thrice weekly for 4 weeks) resulted in a significant increase in tissue mineral density in cortical bone by 5.8% (1416.4±39.27 vs 1338.74±16.56 mgHA/cm 3) and in trabecular bone by 5.2% (1056.52±30.94 vs 1004.13±16.91 mg HA/cm 3) (n=10 in each group, p< 0.05 versus saline-injected controls), as measured by microCT (Figure 1A). To evaluate the capacity of CIB to attenuate EPO mediated bone loss, we administered CIB (300 µg/kg) for 5 consecutive days, to 13-week-old female mice that also received 2 injections of 120U EPO on days 1 and 4. Flow cytometry analysis revealed a 1.8-fold reduction in the number of osteoclast progenitors, defined as Lin -CD11b −CD115 +Ly6C hi, in the EPO + CIB injected mice, compared to the mice injected with EPO alone (n=7 in each group, p< 0.05). Hemoglobin levels and TER119 + bone marrow (BM) erythroid progenitors were similar in both groups. In vitro, EPO administration to BM-derived macrophages (BMDM) enhanced osteoclastogenesis, whereas CIB had an opposite, dose-dependent effect. Combining CIB with EPO inhibited osteoclastogenesis in BMDM, suggesting that CIB overrides the pro-osteoclastogenic effect of EPO (Figure 1B). Conclusions: Our findings highlight the increasing complexity of EPOR signaling in bone and pave the way for clinical translation through potential combination therapy of EPO and CIB in anemic and in cancer patients. Adjunctive administration of CIB may prevent or attenuate bone loss while preserving the erythropoietic actions of EPO. This study was supported by a grant from the Dotan Hemato-oncology Fund, the Cancer Biology Research Center, Tel Aviv University to DN and YG. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Mice overexpressing galectin-8 [gal-8 transgenic (Tg)], a secreted mammalian lectin, exhibit enhanced bone turnover and reduced bone mass, similar to cases of postmenopausal osteoporosis. Here, we show that gal-8 knockout (KO) mice have increased bone mass accrual at a young age but exhibit accelerated bone loss during adulthood. These phenotypes can be attributed to a gal-8-mediated increase in receptor activator of NF-κB ligand (RANKL) expression that promotes osteoclastogenesis, combined with direct inhibition of osteoblast differentiation, evident by reduced bone morphogenetic protein (BMP) signaling, reduced phosphorylation of receptor regulated mothers against decapentaplegic homolog (R-SMAD) and reduced expression of osteoblast differentiation markers osterix, osteocalcin, runt-related transcription factor 2 (RUNX2), dentin matrix acidic phosphoprotein-1 (DMP1), and alkaline phosphatase. At the same time, gal-8 promotes expression of estrogen receptor α (ESR1). Accordingly, the rate of bone loss is accelerated in ovariectomized, estrogen-deficient gal-8 Tg mice, whereas gal-8 KO mice, having low levels of ESR1, are refractory to ovariectomy. Finally, gal-8 mRNA positively correlates with the mRNA levels of osteoclastogenic markers RANKL, tartrate-resistant acid phosphatase, and cathepsin K in human femurs. Collectively, these findings identify gal-8 as a new physiologic player in the regulation of bone mass.-Vinik, Y., Shatz-Azoulay, H., Hiram-Bab, S., Kandel, L., Gabet, Y., Rivkin, G., Zick, Y. Ablation of the mammalian lectin galectin-8 induces bone defects in mice.

The inbred mouse strain C57BL/6 is commonly used for the generation of transgenic mouse and is a well established strain in bone research. Different vendors supply different substrains of C57BL/6J as wild-type animals when genetic drift did not incur any noticeable phenotype. However, we sporadically observed drastic differences in the bone phenotype of "WT" C57BL/6J mice originating from different labs and speculated that these variations are attributable, at least in part, to the variation between C57BL/6J substrains, which is often overlooked. C57BL/6J-OlaHsd is a commonly used substrain that despite a well defined deletion in the alpha-synuclein (Snca) and multimerin-1 (Mmrn1) genes, was reported to display no obvious phenotype and is used as WT control. Here, we compared the bone phenotype of C57BL/6J-OlaHsd (6J-OLA) to C57BL/6J-RccHsd (6J-RCC) and to the original C57BL/6J (6J-JAX). Using μCT analysis, we found that 6J-OLA mice display a significantly lower trabecular bone mass compared to 6J-RCC and 6J-JAX. PCR analysis revealed that both the Snca and Mmrn1 genes are expressed in bone tissue of 6J-RCC animals but not of 6J-OLA mutants, suggesting either one or both genes play a role in bone metabolism. In vitro analysis demonstrated increase in osteoclasts number and decreased osteoblast mineralization in cells derived from 6J-OLA compared with 6J-RCC. Our data may shed light on unexplained differences in basal bone measurements between different research centers and reiterate the importance of specifying the exact substrain type. In addition, our findings describe the physiological role for Mmrn1 and/or Snca in bone remodeling.

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.

Background and aims: Immunotherapy with anti-CD20-specific antibodies (e.g. rituximab), has become the standard of care for B cell lymphoproliferative disorders and many autoimmune diseases. Despite previously demonstrated role for B cells in bone metabolism, the effect of anti-CD20-mediated B cell depletion on bone mass in human patients has not been thoroughly studied. For example, in rheumatological patients the effect of rituximab on bone mass yielded conflicting results, while in lymphoma patients it has not yet been described. Here, we describe the effect of treatment with anti-CD20-specific antibodies on bone mass in a cohort of patients with follicular lymphoma and propose a plausible mechanism using murine model. Methods: To assess the effect of rituximab on bone mass in lymphoma patients, we retrospectively studied the bone mass in patients with follicular lymphoma (FL) during the maintenance phase of chemoimunotherapy, i.e. when rituximab is administered as monotherapy. FL patients on no maintenance (historical controls) or patients with marginal zone lymphoma were include as a control group. Cross-sectional X-ray imaging (CT/PET-CT), performed at the completion of the induction phase and 6-12 months thereafter, were used to serially assess bone density. Wild-type female C57BL/6J mice and mouse anti-mouse CD20 antibody (Genentech) were used for the animal experimental system. Murine bone structure was assessed by the microCT method. Immunophenotyping of the bone marrow (BM), spleen and peripheral blood cells was performed. ELISA and "real-time" quantitative PCR were used to measure the levels of key mediators of bone remodeling, e.g. RANKL, OPG and TNFα. Standard osteoclastogenic assay was used to assesses the osteoclastogenic potential of BM cells. Results: Rituximab treatment prevented the decline in bone mass observed in patients who did not receive active maintenance therapy, both in the lumbar spine (-2.6% vs -8%) and femoral head (-0.5% vs -5.1%) (n=12 patients in each group, p Conclusions: While many lymphoma patients may suffer from bone loss due to advanced age and glucocorticoid administration, our data suggest additional favorable effect of anti-CD20 treatment in bone preservation. Importantly, our murine studies indicate that B cell depletion has a direct effect on bone remodeling, primarily by reducing the osteoclastogenic signals, thus potentially diminishing bone resorption. This novel unrecognized effect should be taken into consideration when maintenance treatment is considered. MM and DN contributed equally to this work. Figure Disclosures No relevant conflicts of interest to declare.

Changes in bone matrix composition are frequently found with bone diseases and may be associated with increased fracture risk. Bone is rich in the trace element zinc. Zinc was established to play a significant role in the growth, development, and maintenance of healthy bones; however, the mechanisms underlying zinc effects on the integrity of the skeleton are poorly understood. Here, we show that the zinc receptor (ZnR)/Gpr39 is required for normal bone matrix deposition by osteoblasts. Initial analysis showed that Gpr39-deficient ( Gpr39-/-) mice had weaker bones as a result of altered bone composition. Fourier transform infrared spectroscopy analysis showed high mineral-to-matrix ratios in the bones of Gpr39-/- mice. Histologic analysis showed abnormally high numbers of active osteoblasts but normal osteoclast numbers on the surfaces of bones from Gpr39-/- mice. Furthermore, Gpr39-/- osteoblasts had disorganized matrix deposition in vitro with cultures exhibiting abnormally low collagen and high mineral contents, findings that demonstrate a cell-intrinsic role for ZnR/Gpr39 in these cells. We show that both collagen synthesis and deposition by Gpr39-/- osteoblasts are perturbed. Finally, the expression of the zinc transporter Zip13 and a disintegrin and metalloproteinase with thrombospondin motifs family of zinc-dependent metalloproteases that regulate collagen processing was downregulated in Gpr39-/- osteoblasts. Altogether, our results suggest that zinc sensing by ZnR/Gpr39 affects the expression levels of zinc-dependent enzymes in osteoblasts and regulates collagen processing and deposition.-Jovanovic, M., Schmidt, F. N., Guterman-Ram, G., Khayyeri, H., Hiram-Bab, S., Orenbuch, A., Katchkovsky, S., Aflalo, A., Isaksson, H., Busse, B., Jahn, K., Levaot, N. Perturbed bone composition and integrity with disorganized osteoblast function in zinc receptor/Gpr39-deficient mice.

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.

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 Ly6Chi monocytes from the BM. In a model of acute acetaminophen-induced liver injury, EPO administration increased the recruitment of Ly6Chi 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 Ly6Chi monocytes in response to liver injury.

We showed previously that proliferating human islet-derived de-differentiated cells (DIDs) exhibit many characteristics of mesenchymal stem cells. Dispersed DIDs can be induced by serum deprivation to undergo mesenchymal-to-epithelial transition and aggregate into epithelial cell clusters (ECCs). Conversely, ECCs can be induced to disperse and undergo epithelial-to-mesenchymal transition (EMT) by re-addition of mammalian sera. In this study, we show that platelet-derived growth factor BB (PDGF-BB) mimics and mediates serum-induced ECCs' dispersal accompanied by accumulation of cytoplasmic β-catenin and a decrease in the levels of insulin and glucagon mRNAs. Moreover, we show that PDGF-BB-induced dispersal of ECCs is a more general phenomenon that occurs also with bone marrow mesenchymal stem cells (BM-MSCs) and dermal fibroblasts (DFs). In DIDs, BM-MSCs, and DFs, PDGF decreased the levels of DKK1 mRNA, suggesting involvement of the Wnt signaling pathway. PDGF-BB stimulated a significant increase in S473 phosphorylation of Akt and the PI3K specific inhibitor (PIP828) partially inhibited PDGF-BB-induced ECC dispersal. Lastly, the PDGF-receptor (PDGF-R) antagonist JNJ-10198409 inhibited both PDGF-BB--and serum-induced ECC dispersal. Epidermal growth factor (EGF), which shares most of the PDGF signaling pathway, did not induce dispersal and only weakly stimulated Akt phosphorylation. Our data suggest that PDGF-BB mediates serum-induced DIDs dispersal, correlated with the activation of the PI3K-Akt pathway.

Background and aims: Erythropoietin (EPO) is the key regulator of red blood cell production, commonly used in clinical practice to treat certain forms of anemia. Our studies and those of others have demonstrated that EPO administration induces substantial trabecular bone loss. We proposed that EPO-induced bone loss is partially mediated by subsets of bone marrow (BM) B cells that express EPO-R. Mechanistically, EPO upregulates the surface expression of RANKL by BM B cells and augments B cell-derived osteoclastogenesis in vitro. We showed that the latter is likely mediated by pro-B cells expressing the MCS-F receptor (CD115) and capable of transdifferentiation to osteoclasts (Abstract # 1007, EHA 2017). Here we address the role of B cell-specific EPO-R in EPO-induced bone loss (i.e. at supra-physiological EPO levels). Moreover, we demonstrate, for the first time, the occurrence of B cell-derived osteoclastogenesis in vivo, a finding of critical importance in the field of osteohematology. Methods: In order to trace the B cell lineage from its earliest precursors, we used the MB1-Cre mouse line combined with either the R26R-EYFP or the EPO-Rfl/fl mice for lineage tracing and B cell-specific EPO-R knockdown, respectively. Sequential fluorescence and light microscopy were used for the demonstration of B cell-derived osteoclastogenesis in vivo. Human recombinant EPO was administered in vivo at a dose of 180IU thrice weekly for two weeks. Immunophenotyping of BM B cell populations was assessed by multi-color flow cytometry. Results: Using female MB1-Cre; EPO-Rfl/fl (cKD) mice, we found that B cell-specific EPO-R knockdown attenuated the profound EPO-induced trabecular bone loss in the proximal part of the femoral distal metaphysis (proximal BV/TV 0.034±0.012% vs 0.007±0.003% in the cKD vs control mice, p Using the MB1-Cre;R26R-EYFP murine model for B cell lineage tracing, we could demonstrate that some of the TRAP+/ β3 integrin+ bone lining cells were also positive for EYFP (Figure 2). This demonstrates the B cell origin of some of the osteoclasts in vivo. Conclusions: Our work highlights B cells as an important extra-erythropoietic target of EPO-EPO-R signaling that regulates bone homeostasis and might also indirectly affect EPO-stimulated erythropoietic response. The relevance and the mechanisms of the latter phenomenon merits further investigation. Importantly, we present here, for the first time, histological evidence for B cell-derived osteoclastogenesis in vivo, thus opening novel research avenues. DN and YG Equal contribution Funded by the German Israel Foundation, Grant # 01021017 to YG, DN, MR and BW and by the Israel Science Foundation (ISF) Grant No. 343/17 to DN. Disclosures Mittelman: Novartis: Honoraria, Research Funding, Speakers Bureau.

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.

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.

PANC-1 cells are an established line of human pancreatic adenocarcinoma (PAC). Hui et al,1 Hardikar et al,2 and Wu et al3 reported that PANC-1 cells, when changed from serum-containing growth medium to serum-free medium (SFM), undergo differentiation and start expressing glucagon, somatostatin, and insulin. The initial process involves aggregation to form spherical clusters that morphologically resemble pancreatic islets accompanied by mesenchymal-to-epithelial transition.3,4 We have previously reported that PANC-1 cells express 3 subtypes of proteinase-activated receptors (PARs 1, 2, 3).5 The stimulation of PAR-1 or PAR-2 evokes cytosolic free calcium ([Ca]i) transients and accelerates PANC-1 cell aggregation in SFM.5,6 β-Cells secrete insulin in response to glucose. This signaling mechanism depends on glucose metabolism and requires the functional presence of glucose sensing machinery. The first step is a rapid glucose uptake facilitated by GLUT2, followed by its phosphorylation and entrance into the glycolytic cycle leading to elevation of the ATP/ADP ratio. The increase in the ATP/ADP ratio causes closure of K(ATP) channels resulting in depolarization, activation of L-type voltage-sensitive calcium channels (VSCCs) and a rise in [Ca]i leading to insulin secretion.7 Fully differentiated β cells in islets form a characteristic syncytium that underlies coordinated calcium signaling and insulin secretion.8–10 Indeed, single β cells, which cannot establish intercellular communication via gap junctions, show alterations in secretion and insulin gene expression, which are corrected after restoration of β-cell contacts.11–13 Hence, the emergence of glucose signaling and the existence of intercellular communication are hallmarks of functional β-cell differentiation. The present report addresses the question whether the early aggregation and differentiation of PANC-1 cells is accompanied by the emergence of functional glucose signaling machinery and of intercellular communication, as well as the effect of activation of PAR-2 receptors on these processes. We show that differentiating PANC-1 cell clusters exhibit glucose signaling and intercellular transport of a marker fluorescent probe. The activation of the glucose signaling pathway is modestly enhanced by a low concentration of trypsin.

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

Erythropoietin (EPO) primarily regulates red blood cell formation, and EPO serum levels are in- creased on hypoxic stress (e.g., anemia and altitude). In addition to anemia, recent discoveries suggest new thera- peutic indications for EPO, unrelated to erythropoiesis. We investigated the skeletal role of EPO using several models of overexpression (Tg6 mice) and EPO adminis- tration (intermittent/continuous, high/low doses) in adult C57Bl6 female mice. Using microcomputed tomography, histology,andserummarkers,wefoundthatEPOinduced a32%-61%trabecularbonelosscausedby increasedbone resorption (+60%-88% osteoclast number) and reduced bone formation rate (219to274%; P

Bone turnover is regulated by the coupled actions of osteoblasts, the bone-forming cells, and monocyte-derived osteoclasts, which mediate bone resorption. B cells were also shown to regulate bone metabolism, chiefly via paracrine signals. Depending on their state and/or mode of activation, B cells may inhibit or enhance osteoclastogenesis. In mammals, B cell development and maturation occurs in the bone marrow (BM), spleen, and other peripheral lymphoid tissues. In the BM, Pro-B cells sequentially differentiate into Pre-B and immature B cells. Whether BM B cells can transdifferentiate into osteoclasts remains controversial, since osteoclast differentiation from residual monocytic precursors in the cultures was not excluded in the earlier studies. Osteoclasts and B cells arise from distinct myeloid and lymphoid progenitors, respectively, which are downstream of a common multipotent progenitor cell. Monocyte differentiation into osteoclasts relies on monocyte-macrophage colony-stimulating factor (M-CSF) and the receptor activator of nuclear factor kappa B ligand (RANKL). Here, we investigated the possibility that BM B-cells contribute to bone loss by transdifferentiating into bone-resorbing osteoclasts. We found that B220+CD19+ cells can transdifferentiate into multinucleated tartrate-resistant acid phosphatase (TRAP) positive osteoclasts in the presence of RANKL and M-CSF. Our results show that Pro-B cells (B220+CD19+CD43HighIGM-), but not Pre-B cells (B220+CD19+CD43LowIGM-), nor immature B cells (B220+CD19+CD43-IGM+), could transdifferentiate into osteoclasts (16%±3.7 vs. 0.79%±0.28 and 0.48%±0.13 osteoclasts area, respectively). Moreover, among the Pro-B cells, only those expressing M-CSF receptor (CD115) could transdifferentiate into functional osteoclasts (18%±6.55 vs. 0.11%±0.05 osteoclasts area, respectively, Figure 1A and B). To unequivocally establish the generation of osteoclasts from B-cells, we next utilized a mouse model in which all B cell lineage-derived progenies express EYFP. We found that B cells isolated from BM of CD19-Cre:EYFP mice differentiated into TRAP+ multinucleated osteoclasts that were also positive for EYFP (Figure1C). Erythropoietin (EPO) is a crucial kidney-derived hormone responsible for erythropoiesis. Once thought to act solely on the erythroid compartment to potentiate red blood cell production, it became evident that EPO receptors are also found on the monocytic lineage (monocytes, macrophages and dendritic cells). In that respect, we have reported that EPO directly stimulates bone loss via activation of EPO-R signaling in the monocytic lineage (Hiram-Bab et al., 2015). Here we report that B cells express EPO-R, and that EPO enhances Pro-B cell differentiation into osteoclasts by 70% (p Conceivably, in other scenarios, e.g., sex hormone deficiency, certain hematological cancers, and treatment with anti-CD20, increased B cell lymphocytes contribute to the bone loss phenotype due to transdifferentiation of B-cell precursors into osteoclasts. Taken together, our data suggest a new physio-pathological role for BM B cell precursors in bone metabolism, via their capacity to differentiate into functional osteoclasts, and a possible role for EPO in this process. Figure 1 Osteoclastogenesis in vitro from sorted B cells. (A) TRAP staining of osteoclasts derived from the indicated sorted cells originating from BM (10,000 cells per well) and cultured with M-CSF and RANKL. Left - Pro-B cells expressing CD115 (B220+CD19+CD43HighIgM-CD115+). Right - Pro-B cells not expressing CD115 (B220+CD19+CD43HighIgM-CD115-). Data are mean±SEM of osteoclast area, n=5 mice in each group; *p Disclosures No relevant conflicts of interest to declare.

Background and rationale: Identifying risk factors for bone loss is of utmost importance in the prevention of osteoporotic bone fractures. We and others have recently described the dramatic erythropoietin (EPO)-induced bone loss. Recombinant EPO and its long-acting derivative, darbepoetin alpha (DA), are frequently and interchangeably used in clinical practice for similar indications including diseases (e.g. multiple myeloma) in which bone mass may be compromised already, as a part of the disease process. Here, we examined in a murine model, the dose response to the erythroid stimulating agents (ESA) EPO and DA, in the erythroid and bone compartments. Methods:C57B6/JRcchsd 9-week old female mice were treated for two weeks with either EPO or DA using two dose intensities. EPO and DA were administered subcutaneously thrice or once weekly, respectively, for a total weekly dose of 27IU/mouse or 540IU/mouse, hereafter defined correspondingly as intermediate (ID) and high dose (HD) subgroups. Bone morphometry of the distal femoral metaphysis was assessed using microCT. Flow cytometry was used for immunophenotyping of bone marrow (BM) cells. One-way ANOVA was used to compare variables among the treatment groups. Results: First, we found that ID-DA induced bone loss to a similar extent as ID-EPO as reflected by a significant (p0.05 for EPO versus DA groups). In view of the established role of B cells in bone metabolism, we determined the effects of EPO and DA on BM B cell compartment. HD-EPO but not ID-EPO induced a "maturation arrest"-like state in the bone marrow B cell population, increasing the proportion of B220+/CD19-/CD43high/IgM- pre-pro-B two-fold and B220+/CD19+/CD43high/IgM- pro-B cells 2.8-fold (p Conclusion:Applying clinically comparable dosing regimen, both recombinant EPO and DA exert a similar, dose-dependent bone loss, not directly proportional to the erythroid response. However, DA, in contrast to EPO, spares the preosteoblast and bone marrow B cell populations. These finding suggest a different mechanism of action of these closely related ESA preparations on the skeletal and immune systems thus warranting further exploration. Finally, our findings advocate for initiating and maintaining patients with the lowest possible doses of EPO/DA to minimize untoward skeletal and other extra-erythropoietic effects, while achieving the desired correction of anemia. This study was supported by a grant from the Israel Cancer Association to DN, YG and MM. Disclosures No relevant conflicts of interest to declare.

The negative effect of hypoxia on bone metabolism is well established but its mechanism of action is not fully understood. Hypoxia triggers the production of erythropoietin (EPO), a hormone most recognized for its hematopoietic function. An increasing number of roles unrelated to red blood cell production have been attributed to EPO, many of which are mediated by non-erythroid cells. In light of the controversy on the effect of increased serum levels of EPO on the skeleton, we investigated here the effect of the hormone on bone metabolism using EPO overexpressing (Tg6), and EPO-administered adult mice. In these models, the increase in EPO levels is similar to its physiologic increase at high altitude without the confounding direct effect of hypoxia on bone cells. Using microcomputed tomography, histology and serum markers we found that high EPO levels result in a severe trabecular bone loss (-32 to -61% decrease in bone density in Tg6 and EPO-injected animals, respectively; p EPO has direct stimulatory effects in vitro on both macrophages and preosteoclasts thus coupling immune and skeletal systems, Activation of purified BM-derived macrophages with EPO, led to a 33% increase (p=0.01) in phagocytosis of 5T33 multiple myeloma cells. EPO strongly stimulated osteoclastogenesis (TRAP staining, Figure 1B) and pit resorption (measured in calcium-phosphate-coated wells) in a cell-autonomous manner. Furthermore, our data indicate that EPO receptor (EPO-R) signaling in osteoclast precursors involves the Jak2 and PI3K pathways, but is independent of the MAPK/MEK pathway. In addition to the direct effect of EPO on monocyte derived cells, high EPO also led to a 1.6 fold (p=0.03) increase in the transcript levels of the osteoclastogenic cytokine RANKL in total bone marrow. Notably, the major sources of RANKL are B cells and osteoblasts, and our data indicate that both cell types express EPO-R, suggesting that they are direct targets of EPO. Accordingly we found an EPO-related increase in membrane bound RANKL on B cells (1.8-fold, p=0.0003) and osteoblasts (1.16-fold, p=0.001) isolated from bone marrow (Figure 1C). To test whether EPO-R on osteoblasts also mediates the attenuation of bone formation, we treated isolated osteoblasts with EPO. However, EPO treatment in vitro did not inhibit osteoblast differentiation and activity, as demonstrated by RT-qPCR of marker genes and mineralization assays. Taken together, these findings demonstrate that EPO strongly regulates bone mass by stimulating osteoclastogenesis and attenuating bone formation. The osteoclastogenic action of EPO is mediated both directly by EPO-R on the monocytic lineage and indirectly via EPO-R signaling on B cells and osteoblasts. Regarding bone formation, the negative effect of EPO on bone formation rate in vivo was not reproduced in isolated cells, thus implying the involvement of cells beyond the osteoblastic lineage. We also propose a new mechanism for hypoxia-induced bone loss that involves EPO action on monocytic, B cell and osteoblastic lineages. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Background Multiple myeloma (MM) is characterized by clonal expansion of malignant plasma cells and monoclonal gammopathy. It is associated with various clinical manifestations including bone disease and anemia. Clinical introduction of recombinant human erythropoietin (rHuEPO) and its derivatives has been a breakthrough in treating patients with anemia, mainly those with chronic kidney disease and cancer patients on chemotherapy. EPO was found to have additional biological effects, including potentiation of both humoral and cell-mediated immune responses. Our studies and those of others, have shown that EPO treatment for anemia in MM patients was associated with general improved immunological functions and immune-mediated modulation (suppression) of MM. A role for EPO in bone metabolism was previously suggested, but its effects were often controversial and the underlying mechanisms remain to be resolved. Objectives To determine the immunological and skeletal effects as well as the underlying mechanisms of EPO signaling in the 5T33 MM mouse model. Results EPO administration led to a 50% decrease in the levels of the MM pathological κ light chain while attenuating the decrease in normal IgA. These immunoglobulin changes were accompanied by a 70% elevation (p=0.015) of B220+IGM+IgD- immature B cells in the BM of EPO-treated MM mice, compared to diluent-injected MM mice (9.8%±0.91 versus 5.68%±1.19 of B220-gated cells, respectively). We also found a 45% increase (p=0.01) in the level of BM macrophages (F4/80+CD11b+) following EPO administration in both healthy and MM mice. Moreover, stimulation in vitro of purified BM-derived macrophages with EPO, led to a 33% increase (p=0.01) in phagocytosis of the 5T33 MM cells. Th17 cells play an important role in MM pathobiology. Our data demonstrate that in MM mice, rHuEPO administration induces a 70% decrease (p=0.021) in the expression level of RORγt, a Th17 hallmark transcription factor. These findings were associated with a 4-fold increase (p Bone disease is a major component of MM and is associated with significant morbidity. High-resolution CT analysis of the distal femurs confirmed bone loss in 5T33 MM mice. The femur bone density of 5T33 MM mice was 30% lower (p=0.04) than that of their healthy counterparts (3.1%±0.003 versus 4.4%±0.008, respectively). Unexpectedly, EPO treatment of MM mice, in comparison to diluent-injected MM mice, induced a 35% reduction (p=0.004) in trabecular bone density (3.1%±0.003 and 2%±0.007, respectively) and 41% (p Conclusions We show that in MM, EPO acts as a double-edged sword, by stimulating the immune response against MM on one hand, but also accelerating bone resorption on the other, which further alters the already compromised bone tissue. A better understanding of the osteoimmunological roles of EPO may advocate for the use of rHuEPO along with targeted bone protective treatment in MM patients, to attenuate the anemia and MM progression, while also preventing bone damage. ND-U and SH-B have contributed equally. Supported by the Multiple Myeloma Research Foundation (DN). Disclosures: No relevant conflicts of interest to declare.

Abstract 5013 Erythropoietin (Epo), mainly produced by the adult kidney, is the major hormone that promotes erythropoiesis. As such, clinical introduction of recombinant human Epo (rHuEpo) and its derivatives (Epoetins), has been a breakthrough in treating patients with anemia, mainly those with chronic kidney failure and cancer patients on chemotherapy. In contrast to 'red flags' suggesting possible detrimental effects of Epo in certain cases of cancer, the hormone was found by numerous studies to have beneficial effects such as neuroprotective and cardio-protective effects. Immune-mediated anti-cancer effects were also documented, as exemplified in multiple myeloma (MM) patients and mouse models. These effects, at least in part, are mediated by Epo receptors (EpoRs) present on cells other than the erythroid lineage. In this respect, we have recently identified functional EpoRs in murine bone marrow-derived macrophages. MM is characterized by clonal proliferation of malignant plasma cells that produce a pathological paraprotein, accompanied by a decrease in the levels of the normal immunoglobulins. Typically this disease is associated with lytic bone lesions. In 5T33 MM, an experimental murine model of MM (producing an IgG2bk paraprotein), mice that were treated with rHuEpo, displayed a 50% decrease in the levels of the pathological K light chain. Notably, the decline in the levels of normal IgA, typically observed in the MM mice, was attenuated in the Epo-treated MM mice. Administration of rHuEpo led to increased levels of liver (three fold increase) and splenic (ten fold increase) macrophages as determined by the number of F4/80/CD11b expressing cells. These findings were associated with an increase in the transcript levels of the Th1 cytokine IFN-g in the bone marrow of Epo-treated 5T33 MM mice as compared to diluent-treated 5T33 MM mice. As MM is typically associated with a Th2 response, Epo may have a beneficial effect in MM, by shifting the balance towards a Th1 response. Myeloma-associated bone disease is considered a major cause of morbidity in MM patients. Using high-resolution computed tomography analysis of recovered femurs we confirmed that the 5T33 MM mice indeed exhibit bone disease. Femur trabecular bone density (bone volume/total volume) of 5T33 MM mice was 43% lower (p=0. 04) than that of their healthy control counterparts (3. 1%±0. 003 versus 4. 4%±0. 008, respectively). This decrease in bone density reflects a 38% increase (p=0. 001) in the total femur volume (4. 38mm3±0. 13 and 2. 72mm3±0. 95 in the 5T33 MM mice, compared to that of healthy mice) and no significant change in trabecular bone volume. Unexpectedly, Epo treatment of the MM mice induced a 34% reduction (p=0. 004) in trabecular bone density compared to the diluent-treated MM mice (2%±0. 007 and 3. 1%±0. 003, respectively) and 41% (p=1E-6) reduction in trabecular number (1. 54mm−1±0. 266 and 2. 62mm−1±0. 23, respectively). This was accompanied by a 19% increase (p=0. 006) in trabecular bone thickness in the Epo-treated MM mice (0. 043mm±0. 005 and 0. 036mm±0. 003, respectively), suggesting a mechanical compensatory mechanism in response to the extensive bone loss. Taken together, our data demonstrate that Epo's impact extends well beyond erythropoiesis. The administration of rHuEpo may be considered as an adjunct treatment to specific anti-MM therapy in cases which require elevation of the erythrocyte counts and an enhancement of the immune system. Yet, a bone protection regimen (bisphosphonates?) should be considered. This study is supported by the Multiple Myeloma Research Foundation, to DN. Disclosures: No relevant conflicts of interest to declare.