Cytosolic Phospholipase A2-Mediated ICAM-1 Expression Is Calcium Dependent

Abstract

Background. Some human malignancies such as virus-related hepatocellular cancer arise in a setting of chronic inflammation. Upregulation of ICAM-1 is a seminal late event in malignant transformation following chronic inflammation. Cytosolic phospholipase A2 (cPLA2) is a lipid-mediator activated by inflammatory stimuli, which has been shown to mediate ICAM-1 upregulation. As lipid mediators are known to work via calcium-dependent mechanisms in nearly all mammalian cells, we hypothesize that inflammatory-mediated ICAM-1 upregulation is dependent on both cPLA2 and intracellular calcium.

Materials and methods. HUVEC were chosen as a representative cell line as they emulate hepatic sinusoids and are a well-established cell model. These were grown to confluence in T-25 flasks and stimulated with TNF-α or LPS for 6 h. Additional groups were preincubated with AACOCF3 (a specific cPLA2 inhibitor) or BAPTA A.M. (a specific inhibitor of intracellular Ca2+) prior to being exposed to inflammatory stimuli. ICAM-1 expression was determined by mean fluorescent intensity (MFI) as measured by FITC-labeled moAb to ICAM-1 via FACS. The role of intracellular Ca2+ on cPLA2 activity was determined by thin-layer chromatography. Groups were compared using ANOVA with Scheffe's post hoc analysis; *P < 0.05 vs control, †P < 0.05 vs LPS and TNF-α was considered significant; N ≥ 4 all experimental groups.

Results. Both cPLA2 and Ca2+ inhibition significantly inhibited inflammatory upregulation of ICAM-1. Pretreatment with BAPTA A.M. attenuated HUVEC cPLA2 activity in response to LPS. These findings suggest that appropriate molecular target suppression may prevent malignant degeneration in the presence of chronic inflammation.