RESUMO
Erythropoietin (EPO) acts by binding to erythroid progenitor cells to regulate red blood cell production. While EPO receptor (Epor) expression is highest on erythroid tissue, animal models exhibit EPO activity in nonhematopoietic tissues, mediated, in part, by tissue-specific Epor expression. This review describes the metabolic response in mice to endogenous EPO and EPO treatment associated with glucose metabolism, fat mass accumulation, and inflammation in white adipose tissue and brain during diet-induced obesity and with bone marrow fat and bone remodeling. During high-fat diet-induced obesity, EPO treatment improves glucose tolerance, decreases fat mass accumulation, and shifts white adipose tissue from a pro-inflammatory to an anti-inflammatory state. Fat mass regulation by EPO is sex dimorphic, apparent in males and abrogated by estrogen in females. Cerebral EPO also regulates fat mass and hypothalamus inflammation associated with diet-induced obesity in males and ovariectomized female mice. In bone, EPO contributes to the balance between adipogenesis and osteogenesis in both male and female mice. EPO treatment promotes bone loss mediated via Epor in osteoblasts and reduces bone marrow adipocytes before and independent of change in white adipose tissue fat mass. EPO regulation of bone loss and fat mass is independent of EPO-stimulated erythropoiesis. EPO nonhematopoietic tissue response may relate to the long-term consequences of EPO treatment of anemia in chronic kidney disease and to the alternative treatment of oral hypoxia-inducible factor prolyl hydroxylase inhibitors that increase endogenous EPO production.
Assuntos
Tecido Adiposo/metabolismo , Osso e Ossos/metabolismo , Encéfalo/metabolismo , Eritropoetina/biossíntese , Receptores da Eritropoetina/biossíntese , Animais , Feminino , Masculino , CamundongosRESUMO
Erythropoietin (EPO) is a hypoxia-inducible hormone, classically known to enhance red blood cell production upon binding its receptor (EPOR) present on the surface of the erythroid progenitor cells. EPO and its receptor are also expressed in the central nervous system (CNS), exerting several non-hematopoietic actions. EPO also plays an important role in the control of breathing. In this review, we summarize the known physiological actions of EPO in the neural control of ventilation during postnatal development and at adulthood in rodents under normoxic and hypoxic conditions. Furthermore, we present the developmental expression patterns of EPO and EPORs in the brainstem, and with the use of in situ hybridization (ISH) and immunofluorescence techniques we provide original data showing that EPOR is abundantly present in specific brainstem nuclei associated with central chemosensitivity and control of ventilation in the ventrolateral medulla, mainly on somatostatin negative cells. Thus, we conclude that EPO signaling may act through glutamatergic neuron populations that are the primary source of rhythmic inspiratory excitatory drive. This work underlies the importance of EPO signaling in the central control of ventilation across development and adulthood and provides new insights on the expression of EPOR at the cellular level.
Assuntos
Tronco Encefálico/crescimento & desenvolvimento , Tronco Encefálico/metabolismo , Eritropoetina/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Receptores da Eritropoetina/biossíntese , Mecânica Respiratória/fisiologia , Animais , Animais Recém-Nascidos , Eritropoetina/genética , Humanos , Camundongos , Ventilação Pulmonar/fisiologia , Receptores da Eritropoetina/genéticaRESUMO
Trypanosoma carassii is a flagellated bloodstream parasite of cyprinid fish with pathogenesis manifesting primarily as anemia in experimentally infected fish. This anemia is characterized by decreases in the number of circulating red blood cells (RBCs) during peak parasitemia. We examined changes in the key blood metrics and expression of genes known to be important in the regulation of erythropoiesis. Increasing parasitemia was strongly correlated with an overall decrease in the total number of circulating RBCs. Gene expression of key erythropoiesis regulators (EPO, EPOR, GATA1, Lmo2, and HIFα) and proinflammatory cytokines (IFNγ and TNFα) were measured and their expressions differed from those in fish made anemic by injections of phenylhydrazine (PHZ). Significant upregulation of pro-erythropoietic genes was observed in PHZ-induced anemia, but not during peak parasitic infection. Previously, we reported on functional characterization of goldfish erythropoietin (rgEPO) and its ability to induce survival and differentiation of erythroid progenitor cells in vitro. Treatment of goldfish during the infection with rgEPO reduced the severity of anemia but failed to fully prevent the onset of the anemic state in infected fish. Proinflammatory cytokines have been implicated in the suppression of erythropoiesis during trypanosomiasis, specifically the cytokines TNFα, IFNγ, and IL-1ß. Analysis of key proinflammatory cytokines revealed that mRNA levels of IFNγ and TNFα were upregulated in response to infection, but only TNFα increased in response to PHZ treatment. Synergistic activity of the proinflammatory cytokines may be required to sustain prolonged anemia. These findings provide insight into the relationship between T. carassii and host anemia and suggest that T. carassii may directly or indirectly suppress host erythropoiesis.
Assuntos
Anemia/genética , Citocinas/biossíntese , Eritropoese/genética , Regulação da Expressão Gênica/genética , Carpa Dourada/parasitologia , Parasitemia/patologia , Trypanosoma/classificação , Anemia/parasitologia , Animais , Contagem de Eritrócitos , Eritropoetina/biossíntese , Fator de Transcrição GATA1/biossíntese , Interferon gama/biossíntese , Proteínas com Domínio LIM/biossíntese , Fenil-Hidrazinas/farmacologia , RNA Mensageiro/genética , Receptores da Eritropoetina/biossíntese , Tripanossomíase/patologia , Fator de Necrose Tumoral alfa/biossínteseRESUMO
BACKGROUND: Paraplegia remains a devastating complication of thoracoabdominal aortic intervention. Metabolic stress induces expression of beta common receptor subunit of erythropoietin (EPO) receptor (ßcR) to exert a neuroprotective effect in spinal cord ischemia reperfusion injury (SCIR). Diazoxide (DZ) has been shown to induce ischemic tolerance. We previously reported that DZ upregulated ßcR expression and enhanced the neuroprotective effects of EPO through the upregulation of ßcR. We hypothesize that ßcR expression induced by DZ before ischemia amplifies the antiapoptotic effects of EPO in a murine model of SCIR. METHODS: Experimental groups included phosphate-buffered saline (PBS) pretreatment + PBS immediately before the operation, PBS+EPO, DZ+PBS, DZ+EPO, and sham. Spinal cord ischemia was induced by a 4-minute thoracic aortic cross-clamp. Functional scoring (Basso Mouse Score) was done at 12-hour intervals for 48 hours. Spinal cords were harvested for histologic analysis, and antiapoptotic factors (caspase 3, 8, and 9, B-cell lymphoma-2, and neuroglobin) were evaluated by Western blot analysis. RESULTS: The motor function of DZ+EPO group was significantly preserved compared with all other groups. The levels of cleaved caspase 8 and 3 in DZ+EPO were significantly lower than in the other groups. Mice treated with DZ+EPO had significantly fewer terminal deoxynucleotide transferase-mediated deoxy uridine triphosphate nick-end labeling-positive cells than other groups. CONCLUSIONS: Optimized upregulation of ßcR by DZ can increase the extrinsic antiapoptotic effects of EPO. Better understanding of this synergetic mechanism may serve to help prevent ischemic complications caused by aortic intervention.
Assuntos
Apoptose/efeitos dos fármacos , Diazóxido/farmacologia , Eritropoetina/farmacologia , Receptores da Eritropoetina/efeitos dos fármacos , Isquemia do Cordão Espinal/prevenção & controle , Animais , Eritropoetina/fisiologia , Camundongos , Receptores da Eritropoetina/biossíntese , Regulação para CimaRESUMO
We have previously demonstrated that recombinant human erythropoietin (rHuEpo) is involved in the regulation of the angiogenic response in multiple myeloma (MM) through a direct effect on macrophages and endothelial cells isolated from the bone marrow of patients with MM. The aim of the present study was designed to determine the effects of rHuEpo on cancer-associated fibroblasts (CAFs) from monoclonal gammopathy of undetermined significance (MGUS) and MM patients by means of in vitro and in vivo assays. rHuEpo treatment reduces the expression of mRNA levels of fibroblast activation markers, namely alpha smooth actin (αSMA) and fibroblast activation protein (FAP) in MGUS and MM CAFs, and of pro-inflammatory and pro-angiogenic cytokines, including interleukin (IL)-6 and IL-8, vascular endothelial growth factor-A (VEGF-A), fibroblast growth factor-2 (FGF-2), and hepatocyte growth factor (HGF) in MM CAFs. Moreover, rHuEpo inhibits the proliferative activity of MM CAFs and increased the apoptosis of MGUS and MM CAFs. Overall, these data suggest that rHu-Epo down-regulates CAFs pro-tumorigenic activity. Moreover, these results are not suggestive for a pro-angiogenic activity of rHuEpo on CAFs. In fact, rHuEpo pre-treatment induces a low angiogenic response in vivo in the chorioallantoic membrane (CAM) assay of MGUS and MM CAFs conditioned medium, not comparable to that of a well-known angiogenic cytokine, VEGF-A, tested in the same assay.
Assuntos
Fibroblastos/efeitos dos fármacos , Gamopatia Monoclonal de Significância Indeterminada/patologia , Mieloma Múltiplo/patologia , Actinas/biossíntese , Actinas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Membrana Corioalantoide/irrigação sanguínea , Membrana Corioalantoide/efeitos dos fármacos , Citocinas/biossíntese , Citocinas/genética , Método Duplo-Cego , Endopeptidases , Epoetina alfa , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Gelatinases/biossíntese , Gelatinases/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação , Masculino , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Neovascularização Fisiológica/efeitos dos fármacos , Receptores da Eritropoetina/biossíntese , Receptores da Eritropoetina/genética , Serina Endopeptidases/biossíntese , Serina Endopeptidases/genéticaRESUMO
BACKGROUND: Expression of hypoxia-inducible factor 1α (HIF-1α) reflects the degree of cell hypoxia and its increased expression was found in most neoplasms, their metastasis as well as in some precancerous lesions. OBJECTIVES: The study aimed to investigate the expression HIF-1α in invasive breast cancer with metastasis to lymph nodes in correlation with steroid receptors (ER-estrogen receptor, PR-progesterone receptor), HER2 (human epidermal growth factor receptor 2) and EPO-R (erythropoietin receptor). MATERIAL AND METHODS: A total of 58 cases of invasive breast cancer with metastasis to lymph nodes were assessed for expression of HIF-1α, EPO-R, ER, PR and HER2. RESULTS: In our study, among all invasive breast cancers, 36.2% exhibited HIF-1α expression in the nuclei of neoplastic cells. We also assessed the correlation between histological type of cancer and basic immunohistochemical profile that included HIF-1α expression and statistical significance was noted only in the case of PR-/ER-/HER2-/ /HIF-1α-/ and PR+/ER+/HER2-/HIF-1α-/ (p = 0.028 and p = 0.008, respectively). However, only in the case of the PR+/ER+/HER2-/HIF-1α+/ immunohistochemical profile and histological grading did we note a statistical significance (p = 0.006). Expression of HIF-1α was most often noted in cancers exhibiting expression of HER2 protein (57.14%). Our study also assessed the relationship between the expression of HIF-1α in invasive breast cancers and the expression of EPO-R and areas of necrosis, demonstrating a statistically significant dependence (p = 0.003). CONCLUSIONS: Expression of HIF-1α was more often noted in invasive HER2+ cancers characterized by high degree of aggressiveness and poorer prognosis, which might suggest that presence of HIF-1α protein expression in HER2+ cancers could be an additional prognostic factor, the frequent occurrence of the phenotype of HIF-1α and EPO-R in cancers invasive HER2 +, in the absence of ER and PR, may suggest that HIF-1α and EPO-R may be an indicator of the aggressiveness of invasive breast cancers, indicating the need for a specific forms of treatment in this group of patients.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias da Mama/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Receptor ErbB-2/biossíntese , Adulto , Idoso , Neoplasias da Mama/metabolismo , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/análise , Imuno-Histoquímica , Metástase Linfática , Pessoa de Meia-Idade , Receptor ErbB-2/análise , Receptores da Eritropoetina/análise , Receptores da Eritropoetina/biossíntese , Receptores de Estrogênio/análise , Receptores de Estrogênio/biossíntese , Receptores de Progesterona/análise , Receptores de Progesterona/biossínteseRESUMO
Recombinant human erythropoietin (rhEPO) is used in breast and ovarian cancer patients to alleviate cancer- and chemotherapy-related anemia. Some clinical trials have reported that rhEPO may adversely impact survival and increase the risk of thrombovascular events in patients with breast cancer but not with ovarian cancer. The latter may potentially benefit the most from rhEPO treatment due to the nephrotoxic and myelosuppresive effects of standard platinum-based chemotherapy used in ovarian cancer disease. However, over the last decade the preclinical data have revealed that EPO is not only the principal growth factor and the hormone which regulates erythropoiesis, but also a cytokine with a pleiotropic activity which also can affect cancer cells. EPO can stimulate survival, ability to form metastases and drug resistance not only in continuous breast- and ovarian cancer cell lines but also in breast cancer stem-like cells. EPO receptor (EPOR) can also be constitutively active in both these cancers and, in breast cancer cells, may act in an interaction with estrogen receptor (ER) and epidermal growth factor receptor-2 (HER-2). EPOR, by an EPO-independent mechanism, promotes proliferation of breast cancer cells in cooperation with estrogen receptor, resulting in decreased effectiveness of tamoxifen treatment. In another interaction, as a result of the molecular antagonism between EPOR and HER2, rhEPO protects breast cancer cells against trastuzumab. Both clinical and preclinical evidence strongly suggest the urgent need to reevaluate the traditional use of rhEPO in the oncology setting.
Assuntos
Antineoplásicos/efeitos adversos , Neoplasias da Mama/tratamento farmacológico , Resistência a Medicamentos , Eritropoetina/efeitos adversos , Neoplasias Ovarianas/tratamento farmacológico , Antineoplásicos/administração & dosagem , Neoplasias da Mama/metabolismo , Eritropoetina/administração & dosagem , Feminino , Humanos , Neoplasias Ovarianas/metabolismo , Receptores da Eritropoetina/biossíntese , Proteínas Recombinantes/efeitos adversos , Transdução de Sinais/efeitos dos fármacos , Doenças Vasculares/induzido quimicamenteRESUMO
The erythropoietin receptor (EpoR) is expressed by cells from the erythroid lineage; however, evidence has accumulated that it is also expressed by some solid tumors. This is an important observation, because recombinant erythropoietin (EPO) is employed in cancer patients to treat anemia related to chemo/radiotherapy. In our studies we employed eight rhabdomyosarcoma (RMS) cell lines (three alveolar-type RMS cell lines and five embrional-type RMS cell lines), and mRNA samples obtained from positive, PAX7-FOXO1-positive, and fusion-negative RMS patient samples. Expression of EpoR was evaluated by RT-PCR, gene array and FACS. The functionality of EpoR in RMS cell lines was evaluated by chemotaxis, adhesion, and direct cell proliferation assays. In some of the experiments, RMS cells were exposed to vincristine (VCR) in the presence or absence of EPO to test whether EPO may impair the therapeutic effect of VCR. We report for a first time that functional EpoR is expressed in human RMS cell lines as well as by primary tumors from RMS patients. Furthermore, EpoR is detectably expressed in both embryonal and alveolar RMS subtypes. At the functional level, several human RMS cell lines responded to EPO stimulation by enhanced proliferation, chemotaxis, cell adhesion, and phosphorylation of MAPKp42/44 and AKT. Moreover, RMS cells became more resistant to VCR treatment in the presence of EPO. Our findings have important potential clinical implications, indicating that EPO supplementation in RMS patients may have the unwanted side effect of tumor progression.
Assuntos
Eritropoetina/genética , Receptores da Eritropoetina/biossíntese , Rabdomiossarcoma/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Resistencia a Medicamentos Antineoplásicos , Eritropoetina/efeitos adversos , Eritropoetina/uso terapêutico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Fosforilação , RNA Mensageiro/biossíntese , Receptores da Eritropoetina/genética , Rabdomiossarcoma/patologia , Vincristina/administração & dosagemRESUMO
OBJECTIVE: Thalassemia is one of the genetic diseases that cause anemia and ineffective erythropoiesis. Increased levels of several inflammatory cytokines have been reported in ß-thalassemia and might contribute to ineffective erythropoiesis. However, the mechanism by which tumor necrosis factor-alpha (TNF-α) is involved in ineffective erythropoiesis in thalassemic patients remains unclear. The objective of this study is to investigate the effect of TNF-α on the erythropoietin (EPO) and erythropoietin receptor (EPOR) expression involved in proliferation of ß-thalassemia/hemoglobin (Hb) E erythroid progenitor cells compared with cells from healthy subjects. MATERIALS AND METHODS: CD34-positive cells were isolated from heparinized blood by using the EasySep® CD34 selection kit. Cells were then cultured with suitable culture medium in various concentrations of EPO for 14 days. The effect of TNF-α on percent cell viability was analyzed by trypan blue staining. In addition, the percentage of apoptosis and levels of EPOR protein were measured by flow cytometry. RESULTS: Upon EPO treatment, a higher cell number was observed for erythroid progenitor cells from both healthy participants and ß-thalassemia/Hb E patients. However, a reduction of apoptosis was found in EPO-treated cells especially for ß-thalassemia/Hb E patients. Interestingly, TNF-α caused higher levels of cell apoptosis and lower levels of EPOR protein in thalassemic erythroid progenitor cells. CONCLUSION: TNF-α caused a reduction in the level of EPOR protein and EPO-induced erythroid progenitor cell proliferation. It is possible that TNF-α could be involved in the mechanism of ineffective erythropoiesis in ß-thalassemia/Hb E patients.
Assuntos
Células Precursoras Eritroides/efeitos dos fármacos , Eritropoese/efeitos dos fármacos , Eritropoetina/farmacologia , Hemoglobina E , Hemoglobinúria/patologia , Receptores da Eritropoetina/biossíntese , Fator de Necrose Tumoral alfa/farmacologia , Talassemia beta/patologia , Adulto , Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Células Precursoras Eritroides/citologia , Eritropoese/fisiologia , Eritropoetina/sangue , Regulação da Expressão Gênica/efeitos dos fármacos , Hemoglobina E/genética , Hemoglobinúria/genética , Heterozigoto , Humanos , Cultura Primária de Células , Receptores da Eritropoetina/genética , Adulto Jovem , Talassemia beta/genéticaRESUMO
AIMS: Chemotherapy induces anaemia in neuroblastoma patients. Cancer-associated anaemia may be treated with recombinant erythropoietin. However, the potential effects of erythropoietin on neuroblastoma and kidney cells have not been extensively evaluated. The present study was designed to investigate the effect of erythropoietin on the proliferation, and protection against vincristine- and etoposide-induced cell death in neuroblastoma (MSN), and embryonic kidney (HEK 293) cells. MAIN METHODS: The expression of erythropoietin and its receptor in MSN and HEK 293 was analysed by RT-PCR, immunocytochemistry, and Western blotting. The effect of erythropoietin on cell viability and proliferation was evaluated by the MTT assay, and by the Click-iT EdU Alexa Fluor 647 kit, respectively. For the cyto-protective assays, cells were incubated with erythropoietin before etoposide and vincristine treatment. Activation of signalling pathways was studied by Western blotting. KEY FINDINGS: MSN and HEK 293 cells expressed the erythropoietin receptor, but not erythropoietin. Erythropoietin induced proliferation and protection against vincristine and etoposide in MSN cells. HEK 293 cells were not affected by erythropoietin. Erythropoietin showed an anti-apoptotic effect which was dependent on the activation of ERK1/2 and AKT. HEK 293 cells presented constitutively phosphorylated AKT, and showed no activation of ERK1/2 upon erythropoietin stimulation. SIGNIFICANCE: These results indicate that erythropoietin induces proliferation of MSN cells, and that it can ameliorate vincristine- and etoposide-induced apoptosis of these cells. Erythropoietin-mediated neuroprotection was regulated by the combined effect of the ERK1/2 and AKT signalling pathways. Our findings provide further insights into the potential effect of erythropoietin on neuroblastoma cells.
Assuntos
Eritropoetina/farmacologia , Etoposídeo/efeitos adversos , Neuroblastoma/patologia , Substâncias Protetoras/farmacologia , Transdução de Sinais/efeitos dos fármacos , Vincristina/efeitos adversos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Eritropoetina/biossíntese , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HEK293 , Humanos , Rim/citologia , Rim/efeitos dos fármacos , Neuroblastoma/enzimologia , Neuroblastoma/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores da Eritropoetina/biossínteseRESUMO
Hematopoietic lineage commitment is regulated by cytokines and master transcription factors, but it remains unclear how a progenitor cell chooses a lineage in the face of conflicting cues. Through transcript counting in megakaryocyte-erythroid progenitors undergoing erythropoiesis, we show that the expression levels of the pro-erythropoiesis transcription factor EKLF (also known as KLF1) and receptor EpoR are inversely correlated with their pro-megakaryopoiesis counterparts, FLI-1 and TpoR (also known as MPL). Notably, as progenitors commit to the erythrocyte lineage, EpoR is upregulated and TpoR is strongly downregulated, thus boosting the potency of the pro-erythropoiesis cue erythropoietin and effectively eliminating the activity of the pro-megakaryopoiesis cue thrombopoietin. Based on these findings, we propose a new model for exclusive decision making that explicitly incorporates signals from extrinsic cues, and we experimentally confirm a model prediction of temporal changes in transcript noise levels in committing progenitors. Our study suggests that lineage-specific receptor levels can modulate potencies of cues to achieve robust commitment decisions.
Assuntos
Linhagem da Célula/genética , Eritropoese/genética , Células-Tronco Hematopoéticas/metabolismo , Fatores de Transcrição Kruppel-Like/biossíntese , Proteína Proto-Oncogênica c-fli-1/biossíntese , Receptores da Eritropoetina/biossíntese , Receptores de Trombopoetina/biossíntese , Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Fatores de Transcrição Kruppel-Like/genética , Megacariócitos/citologia , Proteína Proto-Oncogênica c-fli-1/genética , Receptores de Trombopoetina/genética , Trombopoetina/genética , Trombopoetina/metabolismoRESUMO
The use of in vitro colony assays in mammals has contributed to identification of erythroid progenitor cells such as burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) progenitors, and serves to examine functions of erythropoietic growth factors like Erythropoietin (Epo) and Kit ligand. Here, we established an in vitro colony-forming assay capable of investigating erythropoiesis in carp (Cyprinus carpio), cloned and functionally characterized recombinant homologous molecules Epo and Kit ligand A (Kitla), and identified three distinct erythroid progenitor cells in carp. Recombinant carp Epo induced the formation of CFU-E-like and BFU-E-like erythroid colonies, expressing erythroid marker genes, ß-globin, epor and gata1. Recombinant carp Kitla alone induced limited colony formation, whereas a combination of Kitla and Epo dramatically enhanced erythroid colony formation and colony cell growth, as well as stimulated the formation of thrombocytic/erythroid colonies expressing not only erythroid markers but also thrombocytic markers, cd41 and c-mpl. Utilizing this colony assay to examine the distribution of distinct erythroid progenitor cells in carp, we demonstrated that carp head and trunk kidney play a primary role in erythropoiesis, while the spleen plays a secondary. Furthermore, we showed that presumably bi-potent thrombocytic/erythroid progenitor cells localize principally in the trunk kidney. Our results indicate that teleost fish possess mechanisms of Epo- and Kitla-dependent erythropoiesis similar to those in other vertebrates, and also help to demonstrate the diversity of erythropoietic sites among vertebrates.
Assuntos
Eritropoese/fisiologia , Eritropoetina/genética , Fator de Células-Tronco/genética , Células-Tronco/citologia , Animais , Carpas , Fator de Transcrição GATA1/biossíntese , Rim/metabolismo , Glicoproteína IIb da Membrana de Plaquetas/biossíntese , Receptores da Eritropoetina/biossíntese , Proteínas Recombinantes/genética , Baço/metabolismo , Trombopoetina/biossíntese , Globinas beta/biossínteseRESUMO
BACKGROUND: Paraplegia remains a devastating complication of aortic surgery, occurring in up to 20% of complex thoracoabdominal repairs. Erythropoietin (EPO) attenuates this injury in models of spinal cord ischemia. Upregulation of the beta-common receptor (ßcR) subunit of the EPO receptor is associated with reduced damage in murine models of neural injury. This receptor activates anti-apoptotic pathways including signaling transducer and activator of transcription 3 (STAT3). We hypothesized that spinal cord ischemia-reperfusion injury upregulates the ßcR subunit with a subsequent increase in activated STAT3. METHODS: Adult male C57/BL6 mice received an intraperitoneal injection of 0.5 mL of EPO (10 U/kg) or 0.9% saline after induction of anesthesia. Spinal cord ischemia was induced through sternotomy and 4-minute thoracic aortic cross-clamp. Sham mice underwent sternotomy without cross-clamp placement. Four groups were studied: ischemic and sham groups, each with and without EPO treatment. After 4 hours of reperfusion, spinal cords were harvested and homogenized. The ßcR subunit expression and STAT3 activation were evaluated by immunoblot. RESULTS: Ischemia reperfusion increased ßcR subunit expression in spinal cords of ischemia + saline and ischemia + EPO mice compared with shams (3.4 ± 1.39 vs 1.31 ± 0.3, p = 0.01 and 3.80 ± 0.58 vs 1.56 ± 0.32, p = 0.01). Additionally, both ischemic groups demonstrated increased STAT3 activation compared with shams (1.35 ± 0.14 vs 1.09 ± 0.07, p = 0.01 and 1.66 ± 0.35 vs 1.08 ± 0.17, p = 0.02). CONCLUSIONS: Ischemia-reperfusion injury induces EPO receptor ßcR subunit expression and early downstream anti-apoptotic signaling through STAT3 activation. Further investigation into the role of the ßcR subunit is warranted to determine tissue protective functions of EPO. Elucidation of mechanisms involved in spinal cord protection is essential for reducing delayed paraplegia.
Assuntos
Receptores da Eritropoetina/biossíntese , Traumatismo por Reperfusão/metabolismo , Isquemia do Cordão Espinal/metabolismo , Medula Espinal/irrigação sanguínea , Regulação para Cima , Animais , Células Cultivadas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator de Transcrição STAT3/fisiologiaRESUMO
As essential mediators of red cell production, erythropoietin (EPO) and its cell surface receptor (EPO receptor [EPOR]) have been intensely studied. Early investigations defined basic mechanisms for hypoxia-inducible factor induction of EPO expression, and within erythroid progenitors EPOR engagement of canonical Janus kinase 2/signal transducer and activator of transcription 5 (JAK2/STAT5), rat sarcoma/mitogen-activated protein kinase/extracellular signal-regulated kinase (RAS/MEK/ERK), and phosphatidylinositol 3-kinase (PI3K) pathways. Contemporary genetic, bioinformatic, and proteomic approaches continue to uncover new clinically relevant modulators of EPO and EPOR expression, and EPO's biological effects. This Spotlight review highlights such factors and their emerging roles during erythropoiesis and anemia.
Assuntos
Células Precursoras Eritroides/metabolismo , Eritropoese/fisiologia , Eritropoetina/biossíntese , Regulação da Expressão Gênica/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Receptores da Eritropoetina/biossíntese , Anemia/genética , Anemia/metabolismo , Animais , Células Precursoras Eritroides/citologia , Eritropoetina/genética , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Ratos , Receptores da Eritropoetina/genética , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
Erythropoietin (Epo) and its receptor (EpoR) are required for the regulation of erythropoiesis. Epo binds to the EpoR homodimer on the surface of erythroid progenitors and erythroblasts, and positions the intracellular domains of the homodimer to be in close proximity with each other. This conformational change is sufficient for the initiation of Epo-EpoR signal transduction. Here, we established a system of chemically regulated erythropoiesis in transgenic mice expressing a modified EpoR intracellular domain (amino acids 247-406) in which dimerization is induced using a specific compound (chemical inducer of dimerization, CID). Erythropoiesis is reversibly induced by oral administration of the CID to the transgenic mice. Because transgene expression is limited to hematopoietic cells by the Gata1 gene regulatory region, the effect of the CID is limited to erythropoiesis without adverse effects. Additionally, we show that the 160 amino acid sequence is the minimal essential domain of EpoR for intracellular signaling of chemically inducible erythropoiesis in vivo. We propose that the CID-dependent dimerization system combined with the EpoR intracellular domain and the Gata1 gene regulatory region generates a novel peroral strategy for the treatment of anemia.
Assuntos
Anemia/genética , Eritropoese/genética , Eritropoetina/genética , Fator de Transcrição GATA1/genética , Receptores da Eritropoetina/genética , Sequência de Aminoácidos/genética , Anemia/tratamento farmacológico , Animais , Eritropoese/efeitos dos fármacos , Eritropoetina/biossíntese , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Multimerização Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Receptores da Eritropoetina/biossíntese , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Tacrolimo/administração & dosagem , Tacrolimo/análogos & derivadosRESUMO
Recently it was found that the erythropoietin receptor (EpoR) is expressed on innate immune cells, such as dendritic cells and macrophages. We found that murine bone marrow-derived mast cells express the EpoR and that its expression is increased under hypoxic conditions. Interestingly, Epo stimulation of the cells did not activate signal transducer and activator of transcription molecules, nor did we find differences in the expression of typical STAT-dependent genes, the proliferation rate, and the ability to differentiate or to protect the cells from apoptosis. Instead, we demonstrate that stimulation of mast cells with Epo leads to phosphorylation of the receptor tyrosine kinase c-kit. We hypothesize that this is due to the formation of a receptor complex between the EpoR and c-kit. The common beta chain of the IL-3 receptor family, which was described as part of the tissue protective receptor (TPR) on other non-erythroid cells, however is not activated. To investigate whether the EpoR/c-kit complex has tissue protective properties, cells were treated with the Toll-like receptor ligand LPS. Combined Epo and LPS treatment downregulated the inflammatory response of the cells as detected by a decrease in IL-6 and TNF-α secretion.
Assuntos
Eritropoetina/farmacologia , Mediadores da Inflamação/farmacologia , Inflamação/imunologia , Mastócitos/imunologia , Receptores da Eritropoetina/imunologia , Animais , Antibióticos Antineoplásicos/farmacologia , Hipóxia Celular , Células Cultivadas , Doxorrubicina/farmacologia , Feminino , Interleucina-6/biossíntese , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Complexos Multiproteicos , Fosforilação , Proteínas Proto-Oncogênicas c-kit/metabolismo , Receptores da Eritropoetina/biossíntese , Fator de Transcrição STAT3/biossíntese , Fator de Transcrição STAT5/biossíntese , Transdução de Sinais/imunologia , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Hemoglobin is produced mainly in erythroid cells. However, it has been reported in non-erythroid cells of human and rodents. We have shown previously that neuroglobin, cytoglobin and hemoglobin are expressed in human glioblastoma multiforme (GBM) cells. We sought to determine whether hemoglobin expression is upregulated by hypoxia, and whether its expression is restricted to the cancer stem cell populations in different GBM cell lines or GBM brain tumor initiating cells (BTICs). Flow cytometry, magnetic cell sorting and qRT-PCR were used to examine the hypoxic upregulation of hemoglobins as well as erythropoietin (EPO) and erythropoietin receptor (EPOR) in GBM cell lines (M006x, M059J, M059K, U87R and U87T) and GBM-BTICs. The data showed significantly increased expression in globins (α, ß, γ, δ, ζ and ε), EPO and EPOR mRNA levels under hypoxia. Globin expression is not limited to the stem cell populations or GBM-BTICs but is a property of the entire GBM population. We assumed that the total expression of mRNA of different normalized globins (α, ß, γ, δ, ζ and ε) at different timepoints for the same cell line is 100%. Under aerobic conditions, ε globin was predominantly expressed, and then decreased gradually with increasing time in hypoxia. This was coupled to a concomitant increase in α and γ globins. Our findings suggest that hypoxic upregulation of hemoglobin expression in GBM cells may be a part of a repertoire of active defence and adaptation mechanisms enabling these cells to acquire resistance to aggressive multimodality treatments of chemotherapy and radiotherapy. New therapeutic strategies to interfere with hemoglobin expression or function in GBM cells are required.
Assuntos
Neoplasias Encefálicas/genética , Hipóxia Celular/genética , Glioblastoma/genética , Hemoglobinas/biossíntese , Neoplasias Encefálicas/patologia , Eritropoetina/biossíntese , Regulação Neoplásica da Expressão Gênica , Glioblastoma/patologia , Hemoglobinas/genética , Humanos , Células-Tronco Neoplásicas , Receptores da Eritropoetina/biossínteseRESUMO
Testing for the presence of specific cell-surface receptors (such as EGFR or HER2) on tumor cells is an integral part of cancer care in terms of treatment decisions and prognosis. Understanding the strengths and limitations of these tests is important because inaccurate results may occur if procedures designed to prevent false-negative or false-positive outcomes are not employed. This review discusses tests commonly used to identify and characterize cell-surface receptors, such as the erythropoietin receptor (EpoR). First, a summary is provided on the biology of the Epo/EpoR system, describing how EpoR is expressed on erythrocytic progenitors and precursors in the bone marrow where it mediates red blood cell production in response to Epo. Second, studies are described that investigated whether erythropoiesis-stimulating agents could stimulate tumor progression in cancer patients and whether EpoR is expressed and functional on tumor cells or on endothelial cells. The methods used in these studies included immunohistochemistry, Northern blotting, Western blotting, and binding assays. This review summarizes the strengths and limitations of these methods. Critically analyzing data from tests for cell-surface receptors such as EpoR requires understanding the techniques utilized and demonstrating that results are consistent with current knowledge about receptor biology.
Assuntos
Northern Blotting/métodos , Western Blotting/métodos , Medula Óssea/metabolismo , Células Eritroides/metabolismo , Imuno-Histoquímica/métodos , Receptores da Eritropoetina/biossíntese , Animais , Células Eritroides/citologia , Regulação da Expressão Gênica/fisiologia , Humanos , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptores da Eritropoetina/genéticaRESUMO
Erythropoietin (Epo) is the crucial cytokine regulator of red blood cell production, and recombinant human erythropoietin (rHuEpo) is widely used in clinical practice for the treatment of anemia, primarily in kidney disease and in cancer. Increasing evidence suggests several biological roles for Epo and its receptor, Epo-R, unrelated to erythropoiesis, including angiogenesis. Epo-R has been found expressed in various non-haematopoietic cells and tissues, and in cancer cells. Here, we detected the expression of Epo-R in bone marrow-derived macrophages (BMMAs) from multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS) patients and assessed whether Epo/Epo-R axis plays a role in MM macrophage-mediated angiogenesis. We found that Epo-R is over-expressed in BMMAs from MM patients with active disease compared to MGUS patients. The treatment of BMMAs with rHuEpo significantly increased the expression and secretion of key pro-angiogenic mediators, such as vascular endothelial growth factor, hepatocyte growth factor and monocyte chemotactic protein (MCP-1/CCL-2), through activation of JAK2/STAT5 and PI3 K/Akt pathways. In addition, the conditioned media harvested from rHuEpo-treated BMMAs enhanced bone marrow-derived endothelial cell migration and capillary morphogenesis in vitro, and induced angiogenesis in the chorioallantoic membrane of chick embryos in vivo. Furthermore, we found an increase in the circulating levels of several pro-angiogenic cytokines in serum of MM patients with anemia under treatment with Epo. Our findings highlight the direct effect of rHuEpo on macrophage-mediated production of pro-angiogenic factors, suggesting that Epo/Epo-R pathway may be involved in the regulation of angiogenic response occurring in MM.
Assuntos
Medula Óssea/irrigação sanguínea , Eritropoetina/farmacologia , Eritropoetina/fisiologia , Macrófagos/fisiologia , Gamopatia Monoclonal de Significância Indeterminada/fisiopatologia , Mieloma Múltiplo/fisiopatologia , Proteínas de Neoplasias/fisiologia , Neovascularização Patológica/fisiopatologia , Receptores da Eritropoetina/fisiologia , Idoso , Androstadienos/farmacologia , Proteínas Angiogênicas/biossíntese , Proteínas Angiogênicas/sangue , Proteínas Angiogênicas/genética , Proteínas Angiogênicas/metabolismo , Animais , Células da Medula Óssea , Capilares/ultraestrutura , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Membrana Corioalantoide/irrigação sanguínea , Cromonas/farmacologia , Meios de Cultivo Condicionados/farmacologia , Citocinas/sangue , Epoetina alfa , Humanos , Macrófagos/metabolismo , Pessoa de Meia-Idade , Morfolinas/farmacologia , Mieloma Múltiplo/sangue , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Receptores da Eritropoetina/antagonistas & inibidores , Receptores da Eritropoetina/biossíntese , Receptores da Eritropoetina/genética , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Regulação para Cima/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/farmacologia , WortmaninaRESUMO
Erythropoiesis occurs in the African clawed frog, Xenopus laevis and is mediated by erythropoietin (xlEPO), a primary regulator of this process. Previously, we have shown that the xlEPO receptor (xlEPOR), which is expressed by erythroid progenitors that respond to xlEPO, is found predominantly in the liver. The aim of the present study was to determine the dynamics of erythropoiesis in the livers of normal and anemic X. laevis by identifying the number and precise location of mature and immature erythrocytes. We quantified mature and immature erythrocyte numbers by o-dianisidine staining or immunohistochemistry and investigated the dynamics of erythropoiesis in normal, acute hemolytic and blood-loss states by in vivo cell proliferation assays with 5-bromo-2'-deoxyuridine (BrdU). We detected 0.12×10(8) xlEPOR(+) BrdU(+) cells in the liver of the normal X. laevis at 24 h after BrdU injection. Frogs presenting with acute hemolytic anemia and pancytopenia show a 10-fold increase in the number of xlEPOR(+)/BrdU(+) cells (approximately 1.30×10(8) cells) in the liver. The xlEPOR(+) cells are found predominantly on the inner wall of hepatic sinusoids. Hematopoietic progenitors that undergo slow cell cycling were also observed in the hepatic sinusoids. This study clarifies the rate of production of mature and immature erythrocytes per day in the liver of X. laevis and the way that these cell numbers change in response to anemia.