Endocytosis of GM-CSF receptor ß is essential for signal transduction regulating mesothelial-macrophage transition.

During Freund's adjuvant induced inflammation rat mesenteric mesothelial cells transdifferentiate into mesenchymal cell. They express macrophage markers, inflammatory cytokines (TGF-ß, TNFα, IL-6), and specific receptors. When primary mesenteric cultures were treated with GM-CSF and/or TGF-ß (in vitro), similar phenotypic and biological changes were induced. It seemed likely that GM-CSF receptor-ligand complex should be internalized to initiate mesothelial-macrophage transition. To follow the intracellular route of GM-CSF receptor ß, we co-localized this receptor with various endocytic markers (Cav-1, EEA1, Rab7, and Rab11a), and carried out detailed immunocytochemical, statistical and biochemical analyses. Since STAT5 is one of the downstream element of GM-CSF signaling, we followed the expression and phosphorylation level of this transcription factor. Our results showed that in mesenteric mesothelial cells GM-CSF receptor ß is internalized by caveolae, delivered into early endosomes where the signaling events occur, STAT5A is phosphorylated by JAK2, and then translocated into the nucleus. When dynamin-dependent endocytosis of GM-CSFR ß is inhibited by dynasore, phosphorylation of STAT5A is not occurred, confirming, that the internalization of receptor ß is indispensable for signal transduction. At the early time of inflammation a significant receptor recycling can be found to the plasma membrane. Later (day 8) the receptor is delivered into late endosomes, indicating that its degradation has already started, and the regeneration of mesothelial cells can start. All of these data strongly support that the internalization of GM-CSF receptor ß is required and essential for signal transduction.

Activation of the ß-common receptor by erythropoietin impairs acetylcholine-mediated vasodilation in mouse mesenteric arterioles.

Clinically, erythropoietin (EPO) is known to increase systemic vascular resistance and arterial blood pressure. However, EPO stimulates the production of the potent vasodilator, nitric oxide (NO), in culture endothelial cells. The mechanism by which EPO causes vasoconstriction despite stimulating NO production may be dependent on its ability to activate two receptor complexes, the homodimeric EPO (EPOR2 ) and the heterodimeric EPOR/ß-common receptor (ßCR). The purpose of this study was to investigate the contribution of each receptor to the vasoactive properties of EPO. First-order, mesenteric arteries were isolated from 16-week-old male C57BL/6 mice, and arterial function was studied in pressure arteriographs. To determine the contribution of each receptor complex, EPO-stimulating peptide (ESP), which binds and activates the heterodimeric EPOR/ßCR complex, and EPO, which activates both receptors, were added to the arteriograph chamber 20 min prior to evaluation of endothelium-dependent (acetylcholine, bradykinin, A23187) and endothelium-independent (sodium nitroprusside) vasodilator responses. Only ACh-induced vasodilation was impaired in arteries pretreated with EPO or ESP. EPO and ESP pretreatment abolished ACh-induced vasodilation by 100% and 60%, respectively. EPO and ESP did not affect endothelium-independent vasodilation by SNP. Additionally, a novel ßCR inhibitory peptide (ßIP), which was computationally developed, prevented the impairment of acetylcholine-induced vasodilation by EPO and ESP, further implicating the EPOR/ßCR complex. Last, pretreatment with either EPO or ESP did not affect vasoconstriction by phenylephrine and KCl. Taken together, these findings suggest that acute activation of the heterodimeric EPOR/ßCR in endothelial cells leads to a selective impairment of ACh-mediated vasodilator response in mouse mesenteric resistance arteries.

The therapeutic potential of erythropoiesis-stimulating agents for tissue protection: a tale of two receptors.

Erythropoietin (EPO) is a well-known therapeutic protein employed widely in the treatment of anemia. Over the past decade, abundant evidence has shown that in addition to its systemic role in the regulation of plasma pO(2) by modulating erythrocyte numbers, EPO is also a cytoprotective molecule made locally in response to injury or metabolic stress. Many studies have shown beneficial effects of EPO administration in reducing damage caused by ischemia-reperfusion, trauma, cytotoxicity, infection and inflammation in a variety of organs and tissues. Notably, the receptor mediating the nonerythropoietic effects of EPO differs from the one responsible for hematopoiesis. The tissue-protective receptor exhibits a lower affinity for EPO and is a heteromer consisting of EPO receptor monomers in association with the common receptor that is also employed by granulocyte macrophage colony-stimulating factor, interleukin 3, and interleukin 5. This heteromeric receptor is expressed immediately following injury, whereas EPO production is delayed. Thus, early administration of EPO can dramatically reduce the deleterious components of the local inflammatory cascade. However, a high dose of EPO is required and this also stimulates the bone marrow to produce highly reactive platelets and activates the vascular endothelium into a prothrombotic state. To circumvent these undesirable effects, the EPO molecule has been successfully altered to selectively eliminate erythropoietic and prothrombotic potencies, while preserving tissue-protective activities. Very recently, small peptide mimetics have been developed that recapitulate the tissue-protective activities of EPO. Nonerythropoietic tissue-protective molecules hold high promise in a wide variety of acute and chronic diseases.

Does erythropoietin have a dark side? Epo signaling and cancer cells.

Erythropoietin (Epo) stimulates red blood cell production by docking with its cognate receptor on the erythroid progenitor cell and triggering an array of signaling pathways that inhibit apoptosis and promote cell proliferation and differentiation. In its pharmaceutical forms, epoetin and darbepoetin, Epo is widely used to treat various anemias, including those associated with cancer. The Epo receptor is also expressed by nonhematopoietic cells, including cancer cells, and Epo exhibits a "tissue-protective" effect on nonhematopoietic tissues, possibly mediated through a novel heteroreceptor, blocking apoptosis induced by a variety of insults. The unexpected results of several clinical studies in which Epo was used to treat cancer patients have now raised the question of a potential direct growth-promoting action of Epo on cancer cells.