Thrombopoietin and erythropoietin activate inside-out signaling of integrin and enhance adhesion to immobilized fibronectin in human growth-factor-dependent hematopoietic cells

Abstract

Erythropoietin (EPO) and thrombopoietin (c-MPL ligand; TPO) are structurally similar cytokines and support respectively, the proliferation and differentiation for erythroid and megakaryocytic lineages, as well as more primitive progenitors. We studied the effect of these cytokines on the induction of adhesion of human growth-factor-dependent hematopoietic cells to immobilized fibronectin, which is a main component of the extracellular matrix in the bone marrow. MO7ER cells that are genetically engineered to express human EPO receptor and MO7e cells that express endogenous c-MPL were used. Stimulation with either TPO or EPO induced rapid increases in adhesion of M07ER cells to fibronectin without apparent change of expression of integrins. Experiments with inhibitory monoclonal antibodies (mAbs) demonstrated that CD41, which has been reported to be involved in TPO-induced adhesion of megakaryocytic cells, is not responsible for this enhanced adhesion. Anti-beta 1 integrin mAb inhibited adhesion completely, while inhibition by anti-alpha 4 integrin mAb and anti-alpha 5 integrin mAb was partial. Combination of anti-alpha 4 mAb plus anti-alpha 5 mAb completely abolished adhesion, as did anti-beta 1 mAb, suggesting that the adhesion is mediated by both alpha 4 beta 1 and alpha 5 beta 1 integrins. Experiments using inhibitors suggested that ligand binding followed by activation of intracellular tyrosine kinases along with PI3-kinase activation is required. After stimulation of M07ER cells with either TPO or EPO, fibronectin-attached cells, but not cells in suspension, showed tyrosine phosphorylation of focal adhesion kinase, which plays a central role in integrin-mediated signaling. These data suggest that TPO and EPO might be involved in homing/migration to the bone marrow microenvironment by hematopoietic cells that express corresponding receptors.

An Efficient and Robust Statistical Modeling Approach to Discover Differentially Expressed Genes Using Genomic Expression Profiles

“…Carboxypeptidase (MAX.1) J04970 ‫18.5מ‬ <0.0001 1.0000 1.0000 MAX.1 associated with monocyte to macrophage differentiation and is expressed in AML cells (Rehli et al 1995 (Bundgaard et al 1994), NGAL specific granules are marker for neutrophil maturation (Le Cabec et al 1997). Beta 1 integrin D U33880 ‫21.5מ‬ 0.0021 1.0000 1.0000 TPO up-regulates adhesion of hematopoetic progenitors to fibronectin through activation of integrin alpha4beta1 and alpha5beta1 (Gotoh et al 1997). Sp4 transcription factor X68561 ‫60.5מ‬ 0.0030 1.0000 1.0000 Sp4 not well characterized but closely related to Sp1 (Suske 1999), TPO activates several Sp1-dependent genes during megakaryopoiesis (Wang et al 1999 (Streit et al 1999), close relative thrombospondin 1 is negative regulator of megakaryopoiesis (Chen et al 1997;Touhami et al 1997 (Marlton et al 1995;Mancini et al 2000), CML/ALL translocations identified in 10q (Kagan et al 1989;Aguiar et al 1997;Salvati et al 1999 (Melnick et al 1999), isochromosomes on 17q common (Fioretos et al 1999) Biglycan J04599 ‫71.4מ‬ 0.2119 1.0000 1.0000 Xq28 Translocation found in AML (Weis et al 1985), common in ALL (Stern 1996 Dataset from Golub et al (1999).…”

Sustainedex vivoexpansion of hematopoietic stem cells mediated by thrombopoietin

“…In combination with Flt3͞Flk2 ligand, TPO has been reported to support the expansion of human umbilical cord blood HSC up to 2 ϫ 10 5 -fold (31). In addition, TPO modifies the expression of adhesion receptors on HSC and cell lines, inducing adhesion to stroma and extracellular matrix (32,33). Finally, long-term repopulating HSC express c-mpl (34).…”

Regulation of tyrosine phosphorylation in macrophage phagocytosis and chemotaxis

“…Moreover, phagocytosis of IgG-opsonized particles can be inhibited by small interfering RNA reduction of either DOCK180 or CrkII, which is the predominant Crk isoform in macrophages [48]. Since Syk can also phosphorylate Vav [50] and associate with several adaptor molecules such as Crk [51], it is possible that the ITAM recruited Syk may also activate GEFs, dependent or independent of SFKs.…”