Intracellular protein phosphorylation in adherent U937 monocytes mediated by various culture conditions and fibronectin-derived surface ligands.
Macrophages play a central role in the normal healing process after tissue injury and the host response to foreign objects such as biomaterials. The process leading to macrophage adhesion and activation on protein-adsorbed substrates is complex and unresolved. While the use of primary cells offers clinical relevancy, macrophage cell lines offer unique advantages such as availability and relatively homogeneous phenotype as models to probe the molecular mechanism of cell-surface interaction. Our goal was to better characterize the effect of the culture condition and surface-associated ligands on the extent of U937 adhesion. Tyrosine phosphorylation of intracellular proteins was surveyed as a basis to seek a greater understanding of the molecular mechanism involved in mediating U937 adhesion on various ligand-adsorbed surfaces. U937 viability and adhesion on tissue culture polystyrene (TCPS) increased with (i) increasing serum level, (ii) decreasing tyrosine phosphorylation inhibitor AG18 concentration, or (iii) increasing culture time. The adsorption of various adhesion proteins such as fibronectin and peptide ligands (i.e., RGD, PHSRN) on TCPS did not significantly increase the adherent density of U937 when compared with albumin and PBS ligand controls. However, ligand identity and the presence of phorbol myristate acetate dramatically affected the extent (i.e., increase or decrease) and the identity (i.e., molecular weight) of phosphotyrosine proteins in adherent U937 in a time-dependent manner. The extent and identity of phosphotyrosine proteins did not exhibit a clear AG18 dose dependency, rather the level of tyrosine phosphorylation for a distinct group of proteins was either increased or decreased for a given AG18 concentration.
Chen
Xiuxu
X
School of Pharmacy, University of Wisconsin-Madison, Madison, WI 53705, USA.
Zuckerman
Sean T
ST
Kao
Weiyuan John
WJ
D000970:Antineoplastic Agents / Q000494:pharmacology; D002448:Cell Adhesion / Q000502:physiology; D018929:Cell Culture Techniques; D005353:Fibronectins / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D009000:Monocytes / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011506:Proteins / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism; D020032:Tyrphostins / Q000494:pharmacology; D020298:U937 Cells
D000970:Antineoplastic Agents; D005353:Fibronectins; D008024:Ligands; D011506:Proteins; D020032:Tyrphostins; D013755:Tetradecanoylphorbol Acetate; C060641:tyrphostin A23