Effect of surface-adsorbed proteins and phosphorylation inhibitor AG18 on intracellular protein expression in adherent macrophages.
Macrophages are believed to play an important role in the host inflammatory response to implanted biomaterials. However, the mechanism of macrophage adhesion to protein-adsorbed substrates and the subsequent activation and inflammation is unresolved. Previously the effect of various surface-adsorbed proteins and increasing concentrations of phosphorylation inhibitor AG18 on intracellular protein expression levels in adherent human monocytic cell line U937 was identified using SDS-PAGE and densitometry. The protein ligands and AG18 concentrations up or down regulated the expression of a set of proteins ranging from approximately 200 to approximately 23 kDa. In the present work, HPLC coupled tandem mass spectroscopy (LC/MS) was used to identify proteins in these bands. We hypothesized that key proteins in macrophage adhesion and activation could be identified by observing protein expression resulting from various surface-adsorbed ligands and AG18 concentrations. Increasing concentrations of AG18 down or up regulate protein expression in adherent U937 on PBS-adsorbed TCPS at approximately 52, approximately 42 and approximately 23 kDa. AG18 concentrations had no effect on cells on albumin (Alb)-adsorbed surfaces but regulated different protein expression in adherent U937 on fibronectin (FN)-adsorbed TCPS at 40 and 80 microm AG18. Both Alb and FN regulate distinct sets of proteins in adherent cells as surface-adsorbed ligands. Based on the data from LC/MS, both surface associated ligand and increasing concentrations of AG18 modulate shifts in intracellular signaling.
Zuckerman
Sean T
ST
Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53705, USA.
Kao
Weiyuan John
WJ
D000327:Adsorption; D000818:Animals; D002448:Cell Adhesion / Q000502:physiology*; D002460:Cell Line; D006801:Humans; D008024:Ligands; D008264:Macrophages / Q000166:cytology / Q000502:physiology*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D013499:Surface Properties; D020032:Tyrphostins / Q000378:metabolism*
D008024:Ligands; D011506:Proteins; D020032:Tyrphostins