Erythropoietin signaling in osteoblasts is required for normal bone formation and for bone loss during erythropoietin-stimulated erythropoiesis.

Erythropoietin (EPO) regulates erythropoiesis by binding to erythropoietin receptor (Epor) on erythroid progenitor cells. Epor is also expressed on bone forming osteoblasts and bone loss accompanies EPO-stimulated erythropoiesis in mice. Mice with Epor restricted to erythroid tissue exhibit reduced bone and increased marrow adipocytes; in contrast, transgenic mice (Tg) with osteoblastic-specific deletion of Epor exhibit reduced trabecular bone with age without change in marrow adipocytes. By 12 weeks, male Tg mice had 22.2% and female Tg mice had 29.6% reduced trabecular bone volume (BV) compared to controls. EPO administration (1200 U/kg) for 10 days reduced trabecular bone in control mice but not in Tg mice. There were no differences in numbers of osteoblasts, osteoclasts, and marrow adipocytes in Tg mice, suggesting independence of EPO signaling in mature osteoblasts, osteoclasts, and adipocytes. Female Tg mice had increased number of dying osteocytes and male Tg mice had a trend for more empty lacunae. Osteogenic cultures from Tg mice had reduced differentiation and mineralization with reduced Alpl and Runx2 transcripts. In conclusion, endogenous EPO-Epor signaling in osteoblasts is important in bone remodeling, particularly trabecular bone and endogenous Epor expression in osteoblasts is required for bone loss accompanying EPO-stimulated erythropoiesis.

Erythropoietin-Induced Changes in Bone and Bone Marrow in Mouse Models of Diet-Induced Obesity.

Obesity remodels bone and increases bone marrow adipocytes (BMAT), which negatively regulate hematopoiesis and bone. Reduced BMAT could restore altered hematopoiesis and bone features. We analyzed the potential of erythropoietin (EPO), the cytokine required for erythropoiesis, to inhibit BMAT in C57BL6/J mice fed four weeks of a high-fat diet (HFD). Acute EPO administration markedly decreased BMAT in regular chow diet (RCD) and HFD-fed mice, without affecting whole body fat mass. Micro-CT analysis showed EPO reduced trabecular bone in RCD- and HFD-fed mice, but EPO-treated HFD-fed mice maintained cortical bone mineral density and cortical bone volume, which was reduced on RCD. Despite achieving similar increased hematocrits with BMAT loss in RCD- and HFD-fed mice treated with EPO, decreased bone marrow cellularity was only observed in RCD-fed mice concomitant with an increasing percentage of bone marrow erythroid cells. In contrast, in HFD-fed mice, EPO increased endothelial cells and stromal progenitors with a trend toward the normalization of marrow homeostasis. EPO administration increased c-terminal FGF23 and intact serum FGF23 only in HFD-fed mice. These data demonstrate the distinct EPO responses of bone and marrow in normal and obese states, accompanying EPO-induced loss of BMAT.

Nitric oxide and hypoxia stimulate erythropoietin receptor via MAPK kinase in endothelial cells.

Erythropoietin receptor (EPOR) expression level determines the extent of erythropoietin (EPO) response. Previously we showed that EPOR expression in endothelial cells is increased at low oxygen tension and that EPO stimulation of endothelial cells during hypoxia can increase endothelial nitric oxide (NO) synthase (eNOS) expression and activation as well as NO production. We now observe that while EPO can stimulate NO production, NO in turn can regulate EPOR expression. Human umbilical vein endothelial cells (HUVEC) treated with 10-50 µM of NO donor diethylenetriamine NONOate (DETANO) for 24h showed significant induction of EPOR gene expression at 5% and 2% of oxygen. Also human bone marrow microvascular endothelial cell line (TrHBMEC) cultured at 21 and 2% oxygen with 50 µM DETANO demonstrated a time and oxygen dependent induction of EPOR mRNA expression after 24 and 48 h, particularly at low oxygen tension. EPOR protein was also induced by DETANO at 2% oxygen in TrHBMEC and HUVEC. The activation of signaling pathways by NO donor stimulation appeared to be distinct from EPO stimulation. In reporter gene assays, DETANO treatment of HeLa cells at 2% oxygen increased EPOR promoter activity indicated by a 48% increase in luciferase activity with a 2 kb EPOR promoter fragment and a 71% increase in activity with a minimal EPOR promoter fragment containing 0.2 kb 5'. We found that DETANO activated MAPK kinase in TrHBMEC both in normoxia and hypoxia, while MAPK kinase inhibition showed significant reduction of EPOR mRNA gene expression at low oxygen tension, suggesting MAPK involvement in NO mediated induction of EPOR. Furthermore, DETANO stimulated Akt anti-apoptotic activity after 30 min in normoxia, whereas it inhibited Akt phosphorylation in hypoxia. In contrast, EPO did not significantly increase MAPK activity while EPO stimulated Akt phosphorylation in TrHBMEC in normoxia and hypoxia. These observations provide a new effect of NO on EPOR expression to enhance EPO response in endothelial cells, particularly at low oxygen tensions.

The matricellular protein CCN3 regulates NOTCH1 signalling in chronic myeloid leukaemia.

Deregulated NOTCH1 has been reported in lymphoid leukaemia, although its role in chronic myeloid leukaemia (CML) is not well established. We previously reported BCR-ABL down-regulation of a novel haematopoietic regulator, CCN3, in CML; CCN3 is a non-canonical NOTCH1 ligand. This study characterizes the NOTCH1­CCN3 signalling axis in CML. In K562 cells, BCR-ABL silencing reduced full-length NOTCH1 (NOTCH1-FL) and inhibited the cleavage of NOTCH1 intracellular domain (NOTCH1-ICD), resulting in decreased expression of the NOTCH1 targets c-MYC and HES1. K562 cells stably overexpressing CCN3 (K562/CCN3) or treated with recombinant CCN3(rCCN3) showed a significant reduction in NOTCH1 signalling (> 50% reduction in NOTCH1-ICD, p < 0.05).Gamma secretase inhibitor (GSI), which blocks NOTCH1 signalling, reduced K562/CCN3 colony formation but increased that of K562/control cells. GSI combined with either rCCN3 or imatinib reduced K562 colony formation with enhanced reduction of NOTCH1 signalling observed with combination treatments. We demonstrate an oncogenic role for NOTCH1 in CML and suggest that BCR-ABL disruption of NOTCH1­CCN3 signalling contributes to the pathogenesis of CML.

Erythropoietin action in stress response, tissue maintenance and metabolism.

Erythropoietin (EPO) regulation of red blood cell production and its induction at reduced oxygen tension provides for the important erythropoietic response to ischemic stress. The cloning and production of recombinant human EPO has led to its clinical use in patients with anemia for two and half decades and has facilitated studies of EPO action. Reports of animal and cell models of ischemic stress in vitro and injury suggest potential EPO benefit beyond red blood cell production including vascular endothelial response to increase nitric oxide production, which facilitates oxygen delivery to brain, heart and other non-hematopoietic tissues. This review discusses these and other reports of EPO action beyond red blood cell production, including EPO response affecting metabolism and obesity in animal models. Observations of EPO activity in cell and animal model systems, including mice with tissue specific deletion of EPO receptor (EpoR), suggest the potential for EPO response in metabolism and disease.

Low-Magnitude Mechanical Signals Combined with Zoledronic Acid Reduce Musculoskeletal Weakness and Adiposity in Estrogen-Deprived Mice.

Combination treatment of Low-Intensity Vibration (LIV) with zoledronic acid (ZA) was hypothesized to preserve bone mass and muscle strength while reducing adipose tissue accrual associated with complete estrogen (E 2 )-deprivation in young and skeletally mature mice. Complete E 2 -deprivation (surgical-ovariectomy (OVX) and daily injection of aromatase inhibitor (AI) letrozole) were performed on 8-week-old C57BL/6 female mice for 4 weeks following commencement of LIV administration or control (no LIV), for 28 weeks. Additionally, 16-week-old C57BL/6 female E 2 -deprived mice were administered ±LIV twice daily and supplemented with ±ZA (2.5 ng/kg/week). By week 28, lean tissue mass quantified by dual-energy X-ray absorptiometry was increased in younger OVX/AI+LIV(y) mice, with increased myofiber cross-sectional area of quadratus femorii. Grip strength was greater in OVX/AI+LIV(y) mice than OVX/AI(y) mice. Fat mass remained lower in OVX/AI+LIV(y) mice throughout the experiment compared with OVX/AI(y) mice. OVX/AI+LIV(y) mice exhibited increased glucose tolerance and reduced leptin and free fatty acids than OVX/AI(y) mice. Trabecular bone volume fraction and connectivity density increased in the vertebrae of OVX/AI+LIV(y) mice compared to OVX/AI(y) mice; however, this effect was attenuated in the older cohort of E 2 -deprived mice, specifically in OVX/AI+ZA mice, requiring combined LIV with ZA to increase trabecular bone volume and strength. Similar improvements in cortical bone thickness and cross-sectional area of the femoral mid-diaphysis were observed in OVX/AI+LIV+ZA mice, resulting in greater fracture resistance. Our findings demonstrate that the combination of mechanical signals in the form of LIV and anti-resorptive therapy via ZA improve vertebral trabecular bone and femoral cortical bone, increase lean mass, and reduce adiposity in mice undergoing complete E 2 -deprivation. One Sentence Summary: Low-magnitude mechanical signals with zoledronic acid suppressed bone and muscle loss and adiposity in mice undergoing complete estrogen deprivation. Translational Relevance: Postmenopausal patients with estrogen receptor-positive breast cancer treated with aromatase inhibitors to reduce tumor progression experience deleterious effects to bone and muscle subsequently develop muscle weakness, bone fragility, and adipose tissue accrual. Bisphosphonates (i.e., zoledronic acid) prescribed to inhibit osteoclast-mediated bone resorption are effective in preventing bone loss but may not address the non-skeletal effects of muscle weakness and fat accumulation that contribute to patient morbidity. Mechanical signals, typically delivered to the musculoskeletal system during exercise/physical activity, are integral for maintaining bone and muscle health; however, patients undergoing treatments for breast cancer often experience decreased physical activity which further accelerates musculoskeletal degeneration. Low-magnitude mechanical signals, in the form of low-intensity vibrations, generate dynamic loading forces similar to those derived from skeletal muscle contractility. As an adjuvant to existing treatment strategies, low-intensity vibrations may preserve or rescue diminished bone and muscle degraded by breast cancer treatment.

Zoledronic acid improves bone quality and muscle function in a high bone turnover state.

SUMMARY: Zoledronic acid (ZA) prevents muscle weakness in mice with bone metastases; however, its role in muscle weakness in non-tumor-associated metabolic bone diseases and as an effective treatment modality for the prevention of muscle weakness associated with bone disorders, is unknown. We demonstrate the role of ZA-treatment on bone and muscle using a mouse model of accelerated bone remodeling, which represents the clinical manifestation of non-tumor associated metabolic bone disease. ZA increased bone mass and strength and rescued osteocyte lacunocanalicular organization. Short-term ZA treatment increased muscle mass, whereas prolonged, preventive treatment improved muscle mass and function. In these mice, muscle fiber-type shifted from oxidative to glycolytic and ZA restored normal muscle fiber distribution. By blocking TGFß release from bone, ZA improved muscle function, promoted myoblast differentiation and stabilized Ryanodine Receptor-1 calcium channel. These data demonstrate the beneficial effects of ZA in maintaining bone health and preserving muscle mass and function in a model of metabolic bone disease. Context and significance: TGFß is a bone regulatory molecule which is stored in bone matrix, released during bone remodeling, and must be maintained at an optimal level for the good health of the bone. Excess TGFß causes several bone disorders and skeletal muscle weakness. Reducing excess TGFß release from bone using zoledronic acid in mice not only improved bone volume and strength but also increased muscle mass, and muscle function. Progressive muscle weakness coexists with bone disorders, decreasing quality of life and increasing morbidity and mortality. Currently, there is a critical need for treatments improving muscle mass and function in patients with debilitating weakness. Zoledronic acid's benefit extends beyond bone and could also be useful in treating muscle weakness associated with bone disorders.

Targeting ryanodine receptor type 2 to mitigate chemotherapy-induced neurocognitive impairments in mice.

Chemotherapy-induced cognitive dysfunction (chemobrain) is an important adverse sequela of chemotherapy. Chemobrain has been identified by the National Cancer Institute as a poorly understood problem for which current management or treatment strategies are limited or ineffective. Here, we show that chemotherapy treatment with doxorubicin (DOX) in a breast cancer mouse model induced protein kinase A (PKA) phosphorylation of the neuronal ryanodine receptor/calcium (Ca2+) channel type 2 (RyR2), RyR2 oxidation, RyR2 nitrosylation, RyR2 calstabin2 depletion, and subsequent RyR2 Ca2+ leakiness. Chemotherapy was furthermore associated with abnormalities in brain glucose metabolism and neurocognitive dysfunction in breast cancer mice. RyR2 leakiness and cognitive dysfunction could be ameliorated by treatment with a small molecule Rycal drug (S107). Chemobrain was also found in noncancer mice treated with DOX or methotrexate and 5-fluorouracil and could be prevented by treatment with S107. Genetic ablation of the RyR2 PKA phosphorylation site (RyR2-S2808A) also prevented the development of chemobrain. Chemotherapy increased brain concentrations of the tumor necrosis factor-α and transforming growth factor-ß signaling, suggesting that increased inflammatory signaling might contribute to oxidation-driven biochemical remodeling of RyR2. Proteomics and Gene Ontology analysis indicated that the signaling downstream of chemotherapy-induced leaky RyR2 was linked to the dysregulation of synaptic structure-associated proteins that are involved in neurotransmission. Together, our study points to neuronal Ca2+ dyshomeostasis via leaky RyR2 channels as a potential mechanism contributing to chemobrain, warranting further translational studies.

Erythropoietin treatment and the risk of hip fractures in hemodialysis patients.

Erythropoietin (EPO) is the primary regulator of bone marrow erythropoiesis. Mouse models have provided evidence that EPO also promotes bone remodeling and that EPO-stimulated erythropoiesis is accompanied by bone loss independent of increased red blood cell production. EPO has been used clinically for three decades to treat anemia in end-stage renal disease, and notably, although the incidence of hip fractures decreased in the United States generally after 1990, it rose among hemodialysis patients coincident with the introduction and subsequent dose escalation of EPO treatment. Given this clinical paradox and findings from studies in mice that elevated EPO affects bone health, we examined EPO treatment as a risk factor for fractures in hemodialysis patients. Relationships between EPO treatment and hip fractures were analyzed using United States Renal Data System (USRDS) datasets from 1997 to 2013 and Consolidated Renal Operations in a Web-enabled Network (CROWNWeb) datasets for 2013. Fracture risks for patients treated with <50 units of EPO/kg/week were compared to those receiving higher doses by multivariable Cox regression. Hip fracture rates for 747,832 patients in USRDS datasets (1997-2013) increased from 12.0 per 1000 patient years in 1997 to 18.9 in 2004, then decreased to 13.1 by 2013. Concomitantly, average EPO doses increased from 11,900 units/week in 1997 to 18,300 in 2004, then decreased to 8,800 by 2013. During this time, adjusted hazard ratios for hip fractures with EPO doses of 50-149, 150-299, and ≥ 300 units/kg/week compared to <50 units/kg/week were 1.08 (95% confidence interval [CI], 1.01-1.15), 1.22 (95% CI, 1.14-1.31), and 1.41 (95% CI, 1.31-1.52), respectively. Multivariable analyses of 128,941 patients in CROWNWeb datasets (2013) replicated these findings. This study implicates EPO treatment as an independent risk factor for hip fractures in hemodialysis patients and supports the conclusion that EPO treatment may have contributed to changing trends in fracture incidence for these patients during recent decades. Published 2021. This article is a U.S. Government work and is in the public domain in the USA. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Effects of Erythropoietin in White Adipose Tissue and Bone Microenvironment.

Erythropoietin (EPO) is expressed primarily in fetal liver and adult kidney to stimulate red blood cell production. Erythropoietin receptor expression is not restricted to erythroid progenitor cells, and non-erythroid EPO activity includes immune response and bone remodeling. In bone fracture models, EPO administration promotes bone formation and accelerates bone healing. In contrast, in healthy adult mice, exogenous EPO-stimulated erythropoiesis has been concomitant with bone loss, particularly at high EPO, that may be accompanied by increased osteoclast activation. Other EPO-associated responses include reduced inflammation and loss of fat mass with high-fat diet feeding, especially in male mice. While EPO exhibited a sex-dimorphic response in regulation of fat mass and inflammation in obese mice, EPO-stimulated erythropoiesis as well as EPO-associated bone loss was comparable in males and females. EPO administration in young mice and in obese mice resulted in bone loss without increasing osteoclasts, suggesting an osteoclast-independent mechanism, while loss of endogenous EPO decreased bone development and maintenance. Ossicle formation of bone marrow stromal cell transplants showed that EPO directly regulates the balance between osteogenesis and adipogenesis. Therefore, during development, endogenous EPO contributes to normal bone development and in maintaining the balance between osteogenesis and adipogenesis in bone marrow stromal cells, while EPO treatment in mice increased erythropoiesis, promoted bone loss, decreased bone marrow adipogenesis, and increased osteoclast activity. These observations in mouse models suggest that the most prevalent use of EPO to treat anemia associated with chronic kidney disease may compromise bone health and increase fracture risk, especially at a high dose.

The Many Facets of Erythropoietin Physiologic and Metabolic Response.

In mammals, erythropoietin (EPO), produced in the kidney, is essential for bone marrow erythropoiesis, and hypoxia induction of EPO production provides for the important erythropoietic response to ischemic stress, such as during blood loss and at high altitude. Erythropoietin acts by binding to its cell surface receptor which is expressed at the highest level on erythroid progenitor cells to promote cell survival, proliferation, and differentiation in production of mature red blood cells. In addition to bone marrow erythropoiesis, EPO causes multi-tissue responses associated with erythropoietin receptor (EPOR) expression in non-erythroid cells such neural cells, endothelial cells, and skeletal muscle myoblasts. Animal and cell models of ischemic stress have been useful in elucidating the potential benefit of EPO affecting maintenance and repair of several non-hematopoietic organs including brain, heart and skeletal muscle. Metabolic and glucose homeostasis are affected by endogenous EPO and erythropoietin administration affect, in part via EPOR expression in white adipose tissue. In diet-induced obese mice, EPO is protective for white adipose tissue inflammation and gives rise to a gender specific response in weight control associated with white fat mass accumulation. Erythropoietin regulation of fat mass is masked in female mice due to estrogen production. EPOR is also expressed in bone marrow stromal cells (BMSC) and EPO administration in mice results in reduced bone independent of the increase in hematocrit. Concomitant reduction in bone marrow adipocytes and bone morphogenic protein suggests that high EPO inhibits adipogenesis and osteogenesis. These multi-tissue responses underscore the pleiotropic potential of the EPO response and may contribute to various physiological manifestations accompanying anemia or ischemic response and pharmacological uses of EPO.

Erythropoietin modulates bone marrow stromal cell differentiation.

Erythropoietin is essential for bone marrow erythropoiesis and erythropoietin receptor on non-erythroid cells including bone marrow stromal cells suggests systemic effects of erythropoietin. Tg6 mice with chronic erythropoietin overexpression have a high hematocrit, reduced trabecular and cortical bone and bone marrow adipocytes, and decreased bone morphogenic protein 2 driven ectopic bone and adipocyte formation. Erythropoietin treatment (1 200 IU·kg-1) for 10 days similarly exhibit increased hematocrit, reduced bone and bone marrow adipocytes without increased osteoclasts, and reduced bone morphogenic protein signaling in the bone marrow. Interestingly, endogenous erythropoietin is required for normal differentiation of bone marrow stromal cells to osteoblasts and bone marrow adipocytes. ΔEpoRE mice with erythroid restricted erythropoietin receptor exhibit reduced trabecular bone, increased bone marrow adipocytes, and decreased bone morphogenic protein 2 ectopic bone formation. Erythropoietin treated ΔEpoRE mice achieved hematocrit similar to wild-type mice without reduced bone, suggesting that bone reduction with erythropoietin treatment is associated with non-erythropoietic erythropoietin response. Bone marrow stromal cells from wild-type, Tg6, and ΔEpoRE-mice were transplanted into immunodeficient mice to assess development into a bone/marrow organ. Like endogenous bone formation, Tg6 bone marrow cells exhibited reduced differentiation to bone and adipocytes indicating that high erythropoietin inhibits osteogenesis and adipogenesis, while ΔEpoRE bone marrow cells formed ectopic bones with reduced trabecular regions and increased adipocytes, indicating that loss of erythropoietin signaling favors adipogenesis at the expense of osteogenesis. In summary, endogenous erythropoietin signaling regulates bone marrow stromal cell fate and aberrant erythropoietin levels result in their impaired differentiation.

The NOTCH signaling pathway in normal and malignant blood cell production.

The NOTCH pathway is an evolutionarily conserved signalling network, which is fundamental in regulating developmental processes in invertebrates and vertebrates (Gazave et al. in BMC Evol Biol 9:249, 2009). It regulates self-renewal (Butler et al. in Cell Stem Cell 6:251-264, 2010), differentiation (Auderset et al. in Curr Top Microbiol Immunol 360:115-134, 2012), proliferation (VanDussen et al. in Development 139:488-497, 2012) and apoptosis (Cao et al. in APMIS 120:441-450, 2012) of diverse cell types at various stages of their development. NOTCH signalling governs cell-cell interactions and the outcome of such responses is highly context specific. This makes it impossible to generalize about NOTCH functions as it stimulates survival and differentiation of certain cell types, whereas inhibiting these processes in others (Meier-Stiegen et al. in PLoS One 5:e11481, 2010). NOTCH was first identified in 1914 in Drosophila and was named after the indentations (notches) present in the wings of the mutant flies (Bigas et al. in Int J Dev Biol 54:1175-1188, 2010). Homologs of NOTCH in vertebrates were initially identified in Xenopus (Coffman et al. in Science 249:1438-1441, 1990) and in humans NOTCH was first identified in T-Acute Lymphoblastic Leukaemia (T-ALL) (Ellisen et al. in Cell 66:649-61, 1991). NOTCH signalling is integral in neurogenesis (Mead and Yutzey in Dev Dyn 241:376-389, 2012), myogenesis (Schuster-Gossler et al. in Proc Natl Acad Sci U S A 104:537-542, 2007), haematopoiesis (Bigas et al. in Int J Dev Biol 54:1175-1188, 2010), oogenesis (Xu and Gridley in Genet Res Int 2012:648207, 2012), differentiation of intestinal cells (Okamoto et al. in Am J Physiol Gastrointest Liver Physiol 296:G23-35, 2009) and pancreatic cells (Apelqvist et al. in Nature 400:877-881, 1999). The current review will focus on NOTCH signalling in normal and malignant blood cell production or haematopoiesis.

MicroRNAs 130a/b are regulated by BCR-ABL and downregulate expression of CCN3 in CML.

Chronic Myeloid Leukaemia (CML) is a myeloproliferative disorder characterized by the expression of the oncoprotein, Bcr-Abl kinase. CCN3 normally functions as a negative growth regulator, but it is downregulated in CML, the mechanism of which is not known. MicroRNAs (miRNAs) are small non-coding RNAs, which negatively regulate protein translation by binding to the complimentary sequences of the 3' UTR of messenger RNAs. Deregulated miRNA expression has emerged as a hallmark of cancer. In CML, BCR-ABL upregulates oncogenic miRNAs and downregulates tumour suppressor miRNAs favouring leukaemic transformation. We report here that the downregulation of CCN3 in CML is mediated by BCR-ABL dependent miRNAs. Using the CML cell line K562, we profiled miRNAs, which are BCR-ABL dependent by transfecting K562 cells with anti-BCR-ABL siRNA. MiRNA expression levels were quantified using the Taqman Low Density miRNA array platform. From the miRNA target prediction databases we identified miRNAs that could potentially bind to CCN3 mRNA and reduce expression. Of these, miR-130a, miR-130b, miR-148a, miR-212 and miR-425-5p were significantly reduced on BCR-ABL knockdown, with both miR-130a and miR-130b decreasing the most within 24 h of siRNA treatment. Transfection of mature sequences of miR-130a and miR-130b individually into BCR-ABL negative HL60 cells resulted in a decrease of both CCN3 mRNA and protein. The reduction in CCN3 was greatest with overexpression of miR-130a whereas miR-130b overexpression resulted only in marginal repression of CCN3. This study shows that miRNAs modulate CCN3 expression. Deregulated miRNA expression initiated by BCR-ABL may be one mechanism of downregulating CCN3 whereby leukaemic cells evade negative growth regulation.