促红细胞生成素（EPO）和EPO受体（EPOR）是调节红细胞生成所必需的细胞因子。越来越多证据表明，EPO/EPOR在非造血器官包括心脏、大脑和骨骼的发育和再生中发挥重要作用。但目前人们对EPO/EPOR在骨骼发育和再生中的机制知之甚少。最近一些研究表明，系统性或局部注射EPO可促进骨形成。但是EPO/EPOR在骨骼发育和再生中的作用机制尚不明确。 / 实验发现EPO/EPOR在生长板的前肥大软骨和肥大软骨区富集，且可促进软骨细胞增殖。但利用siRNA技术将软骨细胞EPOR沉默后，软骨细胞增殖会受抑制。随后，我们用阿利新蓝对软骨细胞外基质中的的蛋白聚糖进行染色，发现EPO可促进软骨细胞分化。此外软骨细胞标志基因包括SOX9、SOX5、SOX6、2型胶原蛋白和蛋白聚糖表达上调。实验还发现EPO可促进MSCs增殖。同时EPO可促进上述软骨细胞标志基因的表达增加。当基因沉默EPOR或使用EPO封闭肽后，软骨细胞标志基因的表达降低。以上数据表明， EPO促进软骨细胞增殖和分化的功能至少部分通过其同源受体EPOR介导。 / 我们还探讨了在低氧环境下，EPO对软骨细胞的作用。在低氧环境中， EPO/EPOR和HIF-1α mRNA和蛋白质表达水平均上调，且EPO促进软骨祖细胞集落形成。此结果提示EPO在低氧条件下介导软骨细胞增殖。同时在软骨细胞中标，EPO可激活JAK2和STAT3磷酸化，该结果表明JAK/STAT信号介导软骨细胞的生物学功能。 / 体外内皮细胞出芽实验发现EPO明显促进跖骨表面内皮细胞出芽。在小鼠骨折处局部注射EPO 14天后μCT血管成像发现，EPO可促进骨折处血管生成。体外与体内试验结果同时证实EPO可促进血管生成。 / 在骨折术后第7天和14天，藏红O染色发现EPO促进软骨骨痂形成。在术后第28天，X光和μCT扫描发现，三维重建和定量分析显示EPO促进骨形成，且伴随着骨量和骨面积的增加，以及骨生物力学特性的改善。以上结果表明， EPO可有效促进骨折修复。 / 综上所述， EPO/ EPOR信号调控软骨细胞和MSCs增殖及其软骨细胞分化。EPO还调控软骨细胞在低氧环境下的生物学特性。EPO/ EPOR信号分子有助于骨愈合过程中血管生成和骨形成。因此，EPO/EPOR可作为一个新的治疗靶点促进骨骼修复与再生。 / Erythropoietin (EPO) and EPO receptor (EPOR) are essential cytokine signals regulating erythropoiesis. Growing evidences suggest that EPO/EPOR signaling involves in the development and regeneration of non-hematopoietic organs including heart, brain and bone, et al. Several recent studies indicate that administration of EPO locally or systemically promotes bone formation. However, the underlying mechanisms of EPO/EPOR in skeletal development and regeneration remain unknown. / Our results show that EPO and EPOR are abundantly expressed in the pre-hypertrophic and hypertrophic zone of the growth plates. The proliferation rate of chondro-progenitors is increased following EPO treatment Alcian blue staining for extracellular matrix proteoglycan indicates that EPO promotes the differentiation of chondrocytes. This is accompanied by up-regulated chondrogenic marker genes including SOX9, SOX5, SOX6, type 2 collagen and aggrecan. In a parallel study, the proliferation rate of MSCs is increased following EPO treatment. The mRNA expression of above chondrogenic marker genes is also up-regulated. These effects are eliminated following knockdown of EPOR in chondrocytes by siRNA or treatment with EPO block peptide. These findings indicate that EPO promotes the proliferation and differentiation of primary chondrocytes at least partially mediated by its cognate receptor EPOR. / We next examined the role of EPO in chondrocytes under hypoxia. The mRNA and protein levels of EPO/EPOR and HIF-1α are up-regulated under hypoxia. EPO also enhances the colony forming efficiency of chondro-progenitors under hypoxia. This result suggests that EPO may serve as a mediator to regulate proliferation of chondro-progenitors under hypoxic condition. In addition, we show that EPO up-regulates the phosphorylation states of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) in chondrocytes, suggesting that the function of EPO in chondrocytes is mediated through JAK/STAT signaling. / To address the function of EPO in angiogenesis, we performed metatarsal endothelial sprouting assay. The endothelial sprouting is significantly enhanced in metatarsals treated with EPO. This coincide with our in vivo data that local delivery of EPO increases vascularity of the healing bone at day 14 post-fracture in mice as indicated by micro-CT angiography analysis. / Interestingly, in the mouse fracture model, EPO promotes cartilaginous callus formation at days 7 and 14 post-surgery. This results in accelerated osteogenesis at day 28 post-surgery indexed by the radiographical scoring and micro-CT analysis characterized by increased bone volume and bone surface. This is accompanied by improved biomechanical properties of the healing bone. These results indicate that administration of EPO may serve as an efficient therapy to facilitate bone regenerartion. / In conclusion, EPO/EPOR signal regulates of the proliferation and differentiation of chondrocytes and MSCs to promote chondrogenesis. EPO may also function as a positive mediator in chondrocytes in response to low oxygen tension during chondrogenesis. EPO/EPOR signaling also contributes to angiogenesis and osteogenesis during bone healing. Therefore, EPO/EPOR may serve as a novel therapeutic target to promote skeletal regeneration. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Wan, Lin. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 130-152). / Abstracts also in Chinese. / Wan, Lin.

Erythropoietin (EPO) is the primary cytokine regulator of erythropoiesis. Fundamental to this action is the ability of EPO to bind the EPO receptor (EPO-R), and activate the primary associated tyrosine kinase, JAK2. The critical importance of EPO, EPO-R and JAK2 to erythropoiesis is demonstrated by the fatal embryonic anemia that develops upon EPO, EPO-R or JAK2 deletion. Positive regulation of intracellular signal transduction pathways downstream of EPO-R and JAK2 are well documented, but less is known about negative regulation of EPO-R signaling. Two distinct experimental strategies were utilized to examine a subset of the negative regulatory mechanisms underlying EPO-R signaling and erythropoiesis. Mice deficient in the E3 ubiquitin ligase, Cbl, were generated previously and displayed elevated platelet numbers, expansion of splenic red pulp and splenomegaly, suggesting that Cbl-/- mice have defects in megakaryocyte/erythrocyte progenitors or more committed cells of each lineage. Our studies illustrated that genetic ablation of Cbl resulted in elevated total numbers of Burst Forming Unit-Erythroid and Colony Forming Unit-Erythroid, but decreased bone marrow-derived late erythroblasts. Cbl-deficient late erythroblasts displayed elevated apoptosis, as well as increased expression of Foxo3a and increased mRNA levels of the pro-apoptotic genes, Bim and FasL. These studies implicate Cbl as an important negative regulator of multiple facets of erythroid signaling. The discovery that EPO-R is ubiquitinated and degraded by the proteasome and lysosome led us to examine the role of EPO-R ubiquitination on signal transduction and proliferation. Lysine mutagenesis of EPO-R showed that K348, K388 and K428 were the primary ubiquitin acceptor sites when EPO-R mutants were expressed in HEK 293T cells. BaF3 cells expressing an EPO-R deficient in cytoplasmic lysines displayed diminished EPO-mediated EPO-R, JAK2, PKB and STAT5 phosphorylation and could not proliferate in response to EPO. The membrane proximal lysines of EPO-R, K256 and K276, were necessary for proliferation at physiologic EPO concentrations but were not required at saturating EPO concentrations. Single lysine EPO-R add-back mutants restored signaling and proliferation to BaF3 cells at physiologically elevated EPO concentrations, signifying that EPO-R lysines finely mediate EPO-dependent proliferation and signal transduction. These analyses demonstrate a positive regulatory role for lysines in signal transduction and proliferation.

Erythropoietin (EPO) is the primary cytokine regulator of erythropoiesis. Fundamental to this action is the ability of EPO to bind the EPO receptor (EPO-R), and activate the primary associated tyrosine kinase, JAK2. The critical importance of EPO, EPO-R and JAK2 to erythropoiesis is demonstrated by the fatal embryonic anemia that develops upon EPO, EPO-R or JAK2 deletion. Positive regulation of intracellular signal transduction pathways downstream of EPO-R and JAK2 are well documented, but less is known about negative regulation of EPO-R signaling. Two distinct experimental strategies were utilized to examine a subset of the negative regulatory mechanisms underlying EPO-R signaling and erythropoiesis. Mice deficient in the E3 ubiquitin ligase, Cbl, were generated previously and displayed elevated platelet numbers, expansion of splenic red pulp and splenomegaly, suggesting that Cbl-/- mice have defects in megakaryocyte/erythrocyte progenitors or more committed cells of each lineage. Our studies illustrated that genetic ablation of Cbl resulted in elevated total numbers of Burst Forming Unit-Erythroid and Colony Forming Unit-Erythroid, but decreased bone marrow-derived late erythroblasts. Cbl-deficient late erythroblasts displayed elevated apoptosis, as well as increased expression of Foxo3a and increased mRNA levels of the pro-apoptotic genes, Bim and FasL. These studies implicate Cbl as an important negative regulator of multiple facets of erythroid signaling. The discovery that EPO-R is ubiquitinated and degraded by the proteasome and lysosome led us to examine the role of EPO-R ubiquitination on signal transduction and proliferation. Lysine mutagenesis of EPO-R showed that K348, K388 and K428 were the primary ubiquitin acceptor sites when EPO-R mutants were expressed in HEK 293T cells. BaF3 cells expressing an EPO-R deficient in cytoplasmic lysines displayed diminished EPO-mediated EPO-R, JAK2, PKB and STAT5 phosphorylation and could not proliferate in response to EPO. The membrane proximal lysines of EPO-R, K256 and K276, were necessary for proliferation at physiologic EPO concentrations but were not required at saturating EPO concentrations. Single lysine EPO-R add-back mutants restored signaling and proliferation to BaF3 cells at physiologically elevated EPO concentrations, signifying that EPO-R lysines finely mediate EPO-dependent proliferation and signal transduction. These analyses demonstrate a positive regulatory role for lysines in signal transduction and proliferation.

Thesis supervisor: Peter Veng-Pedersen. Includes bibliographic references (p. 309-322).

Thesis (Ph.D.)--University of Iowa, 2006. / Supervisor: Raymond J. Hohl. Includes bibliographical references (leaves 114-129).

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2007. / Includes bibliographical references (leaves 239-270). Also available in electronic version.

Ischaemia-reperfusion injury (IRI) has been identified as a major contributor to both short and long term kidney transplant failure. Experimental evidence from the literature suggests that Erythropoietin (EPO) is tissue protective, reducing both inflammation and apoptosis following IRI. We performed a randomised, double blind, placebo controlled trial examining the tissue protective effect of high dose EPO (100,000iu over 3 days) in 39 recipients of an extended criteria donor kidney or a non-heart-beating donor kidney. The primary endpoints of the study were difference in plasma and urinary biomarker levels (NGAL, IL-18 and KIM-1) in addition to changes in gene expression. Secondary endpoints included safety, clinical data and differences in metabolomics profiles. There was no difference detected between the treatment groups in terms of biomarkers, gene expression, metabolomics profiling or clinical parameters. No adverse events related to EPO therapy were recorded. In addition, we developed a cell model of kidney transplantation using primary tubulo-epithelial cells and HMEC-1 cells, with which to confirm the protective effects of EPO. Treatment with 50U/ml one hour prior to undergoing cold hypoxia resulted in the maximum degree of tissue protection, as measured using an MTT and an LDH assay. No evidence of EPO toxicity was demonstrated. Tubulo-epithelial cells expressed EPOR mRNA and protein. No CD131 receptor could be demonstrated. In summary, EPO confers tissue protection in a cell model of kidney transplantation but this has not been shown to occur in a clinical trial using high dose EPO in recipients of marginal donor kidneys.

Erythroid progenitor cells of the abnormal clone in polycythemia vera (PV) are capable of colony formation in vitro without the addition of erythropoietin, the regulatory hormone required for normal in vivo and-in vitro erythropoiesis. This property of "erythropoietin-independent" colony formation has been considered a marker for the abnormal clone in PV, although recent studies indicate that not all erythropoietic members of the clone may be capable of exhibiting this abnormal phenotype. The present studies were undertaken to investigate the level of maturation at which establishment of an erythropoietin-independent phenotype might be determined. A series of experiments was performed on the replated progeny of single primitive hemopoietic cells already committed to erythropoiesis (primitive BFU-E). First, conditions were established to maximize the number of erythroid colonies obtainable in secondary assays of replated primary colonies of primitive BFU-E origin. Time course studies and experiments with irradiated peripheral blood "feeder" cells treated in different ways established that results were best when primary colonies were allowed to grow for 9 days prior to replating and when 9 day old feeders stored at 4°C were included in the secondary assay medium. Second, a technique was developed for dividing such colonies between 2 secondary assay cultures. Experiments with normal primary colonies transferred to 2 secondary assays, both containing erythropoietin, showed that the variation between true replicates was random, indicating that the procedure used divided each primary colony equally. Third, it was shown that secondary assays to which no erythropoietin was added failed to support erythroid colony formation by progenitors present in normal 9 day old primary colonies. Finally, the distribution of erythropoietin-dependent and erythropoietin-independent phenotypes in individual colonies derived from primitive BFU-E from 5 patients with. PV was assessed by replating experiments. Most of the replated colonies from PV cultures that yielded erythroid colonies in secondary assays containing 3 units of erythropoietin per ml also produced some erythroid colonies in the paired replicate that contained < 0.01 units of erythropoietin per ml. However, fewer colonies were consistently obtained in the low erythropoietin cultures. These results indicate that in PV, most of the primitive erythroid bursts that generate phenotypically abnormal progeny capable of erythroid colony formation under conditions which are non-permissive for normal cells also produce significant numbers of progeny that are phenotypically normal in this respect. It is concluded that the capacity for erythropoietin-independent growth and maturation exhibited in vitro by terminally differentiating members of the abnormal clone in PV is not commonly fixed at or prior to the primitive BFU-E stage of erythropoietic cell development. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Anaemia frequently accompanies chronic inflammatory diseases like rheumatoid arthritis and cancer. It is postulated to result primarily from the suppression of erythropoiesis by inflammatory cytokines. A contributing factor could be the inhibition of erythropoietin synthesis which may also be mediated by cytokines. Erythropoietin is the hormone which regulates erythropoiesis. The aims of this project were to investigate whether cytokines can indeed suppress erythropoietin production, and to determine whether the erythropoietin response in experimental models of acute and chronic inflammation was appropriate for the associated anaemia. Macrophage-conditioned medium, interleukin-1β, interleukin-6, tumour necrosis factor-α, and neopterin were assayed for inhibition of erythropoietin synthesis by HepG2 cells in culture. All, except neopterin, effected dose-dependent reductions in the secretion of the hormone. Interleukin-1β and tumour necrosis factor-α down-regulated erythropoietin gene transcription, whereas interleukin-6 inhibited a post-transcriptional process. Rats with acute inflammation developed a mild anaemia which evoked an increase in their serum levels of erythropoietin. The serum erythropoietin levels were optimal, since rats with acute inflammation and severe phenylhydrazine-induced anaemia did not have lower levels of the hormone than controls with a similar degree of anaemia, but without acute inflammation. Erythropoietin is, therefore, not an acute phase reactant. Mice with cancer developed a progressive anaemia which was not due to bone marrow invasion by tumour cells. During the first fourteen days after inoculating them with cancer cells, the mice responded by increasing their serum levels of erythropoietin as the anaemia worsened. The erythropoietin response was appropriate when compared to mice with the same degree of phenylhydrazine-induced anaemia. Erythropoietin levels measured in mice with tumours older than fourteen days were significantly lower than those of control mice with the same degree of experimental anaemia. These animals were very cachectic, suggesting that a blunted erythropoietin response may depend on disease activity.

Erythropietin (Epo), acting through its receptor (EpoR), is an essential hemotopietic growth factor that regulates the proliferation, differentiation, and survival of erythroid progenitor cells. Perturbations of Epo/EpoR function cause myeloproliferative disease, such as erythrocytosis, or myelodeficient disease, such as anemia. Therefore, defining the mechanisms by which Epo/EpoR control proliferation and differentiation of erythroid cell lineages attracts interest. Ubiquitin-dependent internalization and degradation is a common regulatory mechanisms affecting signaling from a variety of receptors. Although EpoR has been found to be ubiquitinated, the function of EpoR ubiquitination in the regulation of Epo signaling remains unclear. Therefore, the primary goal of this study was to define the role of EpoR ubiquitination in regulating Epo signaling activities and erythroid cell growth. We showed that EpoR was ubiquitinated in response to ligand stimulation, and that loss of EpoR ubiquitination reduced signaling activity and biological responses to low dosages of Epo. We also identified two EpoR lysines that were the primary targets for ubiquitination, and showed that either ubiquitination site supported the enhanced activities of wild-type-EpoR. Ubiquitination of EpoR was also associated with a change in the endocytic pathway mediating internalization of EpoR. Specifically, constitutive internalization of non-ubiquitinated EpoR was found to depend on dynamin activity, while internalization of ubiquitinated EpoR was dynamin-independent but could be inhibited by disrupting lipid raft microdomains in the plasma membrane. Interestingly, inhibiting internalization of ubiquitinated EpoR (by disrupting lipid rafts) specifically reduced signaling from ubiquitinated receptors without affecting signaling from non-ubiquitinated receptors. Conversely, reducing internalization of non-ubiquitinated EpoR (by inhibiting dynamin) reduced its signaling activity without affecting signaling from ubiquitinated receptors. This strong correlation between EpoR internalization and signaling activity suggests a novel regulatory mechanism in which internalization of EpoR facilitates its signaling activity. In this regard, Epo-induced ubiquitination of EpoR promotes more efficient internalization of ligand-activated receptor and may contribute to enhanced responsiveness to low concentrations of Epo.