MATERIALS AND METHODS HUVEC. HUVECs were obtained from umbilical cords from cesarean sections. The cells were cultured by standard methods in the presence of heparin and endothelial-cell growth supplement (7). They were characterized by their homogeneous and typical cobblestone morphology, factor VIII antigen positivity, and the presence of Weibel-Palade bodies on electron microscopy. HUVECs were used for these studies after three to five passages.For clonal culture to exclude contamination with hematopoietic stem cells, HUVECs (1-5 x 105 cells per ml) were plated in methylcellulose cultures as described (8), with the modification that half of the fetal bovine serum was replaced with human umbilical cord blood serum. Hematopoietic growth factors were added as follows: rhEpo, 2 units/ml; stem-cell factor, 10 pg/ml; granulocyte/macrophage-colonystimulating factor, 200 units/ml; interleukin 3, 200 units/ml; endothelial-cell growth factor, 20 ktg/ml.Preparation of mRNA. After 1-5 x 107 cells were harvested and washed twice with phosphate-buffered saline, RNA was extracted with guanidinium thiocyanate and lauryl sarcosinate (9). mRNA was adsorbed onto oligo(dT)-cellulose columns (Pharmacia) and, after the columns were washed with high-and low-salt solutions, was eluted with 10 mM Tris'HCl buffer containing 1 mM EDTA (pH 7.4) at 650C. Total amount and concentration of mRNA were determined spectrophotometrically and confirmed by agarose gel electrophoresis.

Erythropoietin (Epo) is required for the production of mature red blood cells. The requirement for Epo and its receptor (EpoR) for normal heart development and the response of vascular endothelium and cells of neural origin to Epo provide evidence that the function of Epo as a growth factor or cytokine to protect cells from apoptosis extends beyond the hematopoietic lineage. We now report that the EpoR is expressed on myoblasts and can mediate a biological response of these cells to treatment with Epo. Primary murine satellite cells and myoblast C2C12 cells, both of which express endogenous EpoR, exhibit a proliferative response to Epo and a marked decrease in terminal differentiation to form myotubes. We also observed that Epo stimulation activates Jak2/Stat5 signal transduction and increases cytoplasmic calcium, which is dependent on tyrosine phosphorylation. In erythroid progenitor cells, Epo stimulates induction of transcription factor GATA-1 and EpoR; in C2C12 cells, GATA-3 and EpoR expression are induced. The decrease in differentiation of C2C12 cells is concomitant with an increase in Myf-5 and MyoD expression and inhibition of myogenin induction during differentiation, altering the pattern of expression of the MyoD family of transcription factors during muscle differentiation. These data suggest that, rather than acting in an instructive or specific mode for differentiation, Epo can stimulate proliferation of myoblasts to expand the progenitor population during differentiation and may have a potential role in muscle development or repair.Erythropoietin (Epo) 1 is required for the development and maturation of erythroid cells and acts to stimulate the proliferation and differentiation of erythroid progenitor cells. Mice lacking expression of erythropoietin or its receptor die in utero due to insufficient erythropoiesis in the fetal liver (1). Erythropoietin production can be induced by hypoxia and provides physiologic regulation of the red cell mass. Erythropoietin receptor is a member of the cytokine receptor superfamily characterized by a single transmembrane domain, homology in the extracellular domain that includes a WSXWS motif, and a cytoplasmic domain that does not contain a kinase motif. Binding of erythropoietin to its receptor results in receptor dimerization, increased affinity for Jak2 to the receptor's membrane proximal region, and subsequent phosphorylation of Jak2 and tyrosines on the cytoplasmic region of the receptor (2). As with other members of this superfamily such as thrombopoietin, interleukin-3, granulocyte-macrophage colony-stimulating factor, and prolactin, Jak2 is required for signaling (3, 4) and Jak2 phosphorylation activates Stat5 (5, 6) and other signal transduction pathways. A role for calcium has been implicated in erythropoietin activity. For example, in erythroid progenitor cells, erythropoietin activates an increase in intracellular calcium in a dose-dependent manner mediated via tyrosine phosphorylation of the erythropoietin receptor requiring the cytoplasmic tyrosine 4...

While erythropoietin is the cytokine known that regulates erythropoiesis, erythropoietin receptor (EpoR) expression and associated activity beyond hematopoietic tissue remain uncertain. Here we show that mice with EpoR expression restricted to hematopoietic tissues (Tg) develop obesity and insulin resistance. Tg-mice exhibit a decrease in energy expenditure and an increase in white fat mass and adipocyte number. Conversely, erythropoietin treatment of wild-type mice increases energy expenditure and reduces food intake and fat mass accumulation but showed no effect in body weight of Tg-mice. EpoR is expressed at a high level in white adipose tissue and in the proopiomelanocortin neurons of the hypothalamus. While Epo treatment in wild-type mice induces the expression of the polypeptide hormone precursor gene, proopiomelanocortin, mice lacking EpoR show reduced levels of proopiomelanocortin in the hypothalamus. This study provides the first evidence that mice lacking EpoR in nonhematopoietic tissue become obese and insulin resistant with loss of erythropoietin regulation of energy homeostasis.

Erythropoietin (EPO), a hypoxia-inducible cytokine, is required for survival, proliferation, and differentiation of erythroid progenitor cells. EPO can also stimulate proliferation and angiogenesis of endothelial cells that express EPO receptors (EPORs). In this study we investigated the EPO response of vascular endothelial cells at reduced oxygen tension (5% and 2%), in particular the effect of EPO on nitric oxide (NO) release. Endothelial nitric oxide synthase (eNOS) produces NO, which maintains blood pressure homeostasis and blood flow. We find that EPOR is inducible by EPO in primary human endothelial cells of vein (HUVECs) and artery (HUAECs) and cells from a human bone marrow microvascular endothelial line (TrHBMEC) to a much greater extent at low oxygen tension than in room air. We found a corresponding increase in eNOS expression and NO production in response to EPO during hypoxia. Stimulation of NO production was dose dependent on EPO concentration and was maximal at 5 U/mL. NO activates soluble guanosine cyclase to produce cyclic guanosine monophosphate (cGMP), and we observed that EPO induced cGMP activity. These results suggest that low oxygen tension increases endothelial cell capacity to produce NO in response to EPO by induction of both EPOR and eNOS. This effect of EPO on eNOS may be a physiologically relevant mechanism to counterbalance the hypertensive effects of increased hemoglobin-related NO destruction resulting from hypoxia-induced increased red cell

Protein biogenesis at the endoplasmic reticulum (ER) in eukaryotic cells is monitored by a protein quality control system named ERassociated protein degradation (ERAD). While there has been substantial progress in understanding how ERAD eliminates defective polypeptides generated from erroneous folding, how cells remove nascent chains stalled in the translocon during co-translational protein insertion into the ER is unclear. Here we show that ribosome stalling during protein translocation induces the attachment of UFM1, a ubiquitin-like modifier, to two conserved lysine residues near the COOH-terminus of the 60S ribosomal subunit RPL26 (uL24) at the ER. Strikingly, RPL26 UFMylation enables the degradation of stalled nascent chains, but unlike ERAD or previously established cytosolic ribosome-associated quality control (RQC), which uses proteasome to degrade their client proteins, ribosome UFMylation promotes the targeting of a translocation-arrested ER protein to lysosomes for degradation. RPL26 UFMylation is upregulated during erythroid differentiation to cope with increased secretory flow, and compromising UFMylation impairs protein secretion, and ultimately hemoglobin production. We propose that in metazoan, co-translational protein translocation into the ER is safeguarded by a UFMylation-dependent protein quality control mechanism, which when impaired causes anemia in mice and abnormal neuronal development in humans.