Abstract
BackgroundEph signaling is known to induce contrasting cell behaviors such as promoting and inhibiting cell adhesion/spreading by altering F-actin organization and influencing integrin activities. We have previously demonstrated that EphA2 stimulation by ephrin-A1 promotes cell adhesion through interaction with integrins and integrin ligands in two monocyte/macrophage cell lines. Although mature mononuclear leukocytes express several members of the EphA/ephrin-A subclass, their expression has not been examined in monocytes undergoing during differentiation and maturation.ResultsUsing RT-PCR, we have shown that EphA2, ephrin-A1, and ephrin-A2 expression was upregulated in murine bone marrow mononuclear cells during monocyte maturation. Moreover, EphA2 and EphA4 expression was induced, and ephrin-A4 expression was upregulated, in a human promyelocytic leukemia cell line, HL60, along with monocyte differentiation toward the classical CD14++CD16− monocyte subset. Using RT-PCR and flow cytometry, we have also shown that expression levels of αL, αM, αX, and β2 integrin subunits were upregulated in HL60 cells along with monocyte differentiation while those of α4, α5, α6, and β1 subunits were unchanged. Using a cell attachment stripe assay, we have shown that stimulation by EphA as well as ephrin-A, likely promoted adhesion to an integrin ligand-coated surface in HL60 monocytes. Moreover, EphA and ephrin-A stimulation likely promoted the formation of protrusions in HL60 monocytes.ConclusionsNotably, this study is the first analysis of EphA/ephrin-A expression during monocytic differentiation/maturation and of ephrin-A stimulation affecting monocyte adhesion to an integrin ligand-coated surface. Thus, we propose that monocyte adhesion via integrin activation and the formation of protrusions is likely promoted by stimulation of EphA as well as of ephrin-A.
Highlights
Eph signaling is known to induce contrasting cell behaviors such as promoting and inhibiting cell adhesion/ spreading by altering Filamentous actin (F-actin) organization and influencing integrin activities
EphA and ephrin-A are upregulated in bone marrow mononuclear cells during monocytic maturation Macrophage colony-stimulating factor (M-CSF) induces proliferation and differentiation of bone marrow MNCs into the mononuclear phagocytic lineage, wherein the M-CSF receptor signaling is involved in cell adhesion to extracellular matrices [31]
We examined Nonspecific esterase (NSE)-reactivity and the mRNA expression levels of marker molecules (CD115, a monocyte marker [23, 24], CD34, a myeloid cell marker [25]) in adherent MNCs by semi-quantitative RT-PCR in both groups (MC-1d group: non-adherent MNCs cultured in M-CSF-containing medium which became adherent in one day; MC-5d group: non-adherent MNCs cultured in M-CSF-containing medium which became adherent in two days and were cultured for three more days)
Summary
Eph signaling is known to induce contrasting cell behaviors such as promoting and inhibiting cell adhesion/ spreading by altering F-actin organization and influencing integrin activities. We have previously demonstrated that EphA2 stimulation by ephrin-A1 promotes cell adhesion through interaction with integrins and integrin ligands in two monocyte/ macrophage cell lines. Eph receptors and ephrin ligands are membrane proteins that primarily regulate cell-cell repulsion and adhesion as well as cell adhesion and movement by modulating the organization of the actin cytoskeleton mainly via Rho family GTPases [1]. The Eph receptor tyrosine kinase family has 14 members that are divided into the EphA (A1–A8 and A10) and EphB B6) subclasses based on the sequence similarity of their extracellular domains. The members of these two receptor subclasses promiscuously bind the ligands of the ephrin-A (A1–A5) and -B (B1–B3) classes, respectively. Forward signaling by Eph mainly depends on autophosphorylation and phosphorylation by other tyrosine kinases as well as by the
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