title: Multilevel regulation of IL-6R by IL-6-sIL-6R fusion protein according to the primitiveness of peripheral blood-derived CD133+ cells.
abstract: Interleukin-6 (IL-6) and its soluble receptor (sIL-6R) are major factors for maintenance and expansion of hematopoietic stem cells (HSCs). Sensitivity of HSCs to IL-6 has been previously studied, in part by measuring the expression of IL-6R on the membrane (mIL-6R). Several studies have described the regulation of cell surface expression of IL-6R by several cytokines, but the role of glycoprotein 130 activation has not yet been investigated. In this study, CD133(+) cells were purified from adult peripheral blood and were precultured in the absence or presence of 5-fluorouracil (5-FU) for selection of quiescent HSCs. Cells were cultured with continuous or pulsed stimulations of an IL-6-sIL-6R fusion protein (hyperinterleukin-6 [HIL-6]) to 1) detect mIL-6R by flow cytometry, 2) assess mIL-6R and sIL-6R RNAs by reverse transcription-polymerase chain reaction, 3) measure sIL-6R in supernatants by enzyme-linked immunosorbent assay, 4) analyze cell-cycle status, and 5) perform long-term culture-initiating cell assays. The level of mIL-6R(-) cells was preserved by 5-FU incubation. HIL-6 increased steady-state mIL-6R RNA and expression rate on HSCs, independently of treatment with 5-FU. Enhanced production of sIL-6R was observed with short pulses of HIL-6 on CD133(+) 5-FU-pretreated cells. This overproduction of sIL-6R was abrogated by tumor necrosis factor-alpha protease inhibitor-1, an inhibitor of a disintegrin and metalloprotease proteases, suggesting the shedding of mIL-6R. This phenomenon was mediated through the phosphatidylinositol-3'-kinase pathway and was involved in the maintenance of primitive HSCs. In conclusion, expression and production of IL-6R are tightly regulated and stage specific. We assume that sIL-6R produced by shedding should be involved in autocrine and paracrine loops in the HSC microenvironment.
authors.affiliation: Laboratoire Micro-Environnement et le Renouvellement Cellulaire Intégré, Faculté de Médecine, Pharmacie de Rouen, France. david.campard@etu.univ-rouen.fr
authors.forename: David
authors.initials: D
authors.lastname: Campard
authors.forename: Marc
authors.initials: M
authors.lastname: Vasse
authors.forename: Stefan
authors.initials: S
authors.lastname: Rose-John
authors.forename: Florent
authors.initials: F
authors.lastname: Poyer
authors.forename: Marek
authors.initials: M
authors.lastname: Lamacz
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Vannier
mesh_terms: D000071916:AC133 Antigen; D051722:ADAM Proteins / Q000378:metabolism; D015703:Antigens, CD / Q000096:biosynthesis*; D002453:Cell Cycle / Q000187:drug effects*; D002478:Cells, Cultured; D005786:Gene Expression Regulation; D006023:Glycoproteins / Q000096:biosynthesis*; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D017370:Interleukin-11 / Q000494:pharmacology; D053605:Leukemia Inhibitory Factor / Q000494:pharmacology; D008954:Models, Biological; D010455:Peptides; D019947:Receptors, Interleukin-6 / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012995:Solubility
chemical_list: D000071916:AC133 Antigen; D015703:Antigens, CD; D006023:Glycoproteins; D017370:Interleukin-11; D053605:Leukemia Inhibitory Factor; C000606023:PROM1 protein, human; D010455:Peptides; D019947:Receptors, Interleukin-6; D011993:Recombinant Fusion Proteins; D051722:ADAM Proteins