Phorbol 12-myristate 13-acetate (PMA) Alters Galectin Gene Expression in Cows

Abstract

Galectins (GAL) are β-galactoside binding proteins. The objective of this study was to assess the effect of PMA on the regulation of galectin gene expression in bovine blood and neutrophils. Blood was collected from 3 clinically healthy Holstein cows. Neutrophils were isolated by differential centrifugation. Blood and neutrophils were treated with 10ng/ml of PMA or maintained in phosphate-buffered saline for 30 minutes at 370 C. Pooled total RNA were transcribed to cDNA for real-time PCR. Specific primers for bovine LGALS -1, -2, -3, -4, -7, -8, -9, -11, -12, and their binding proteins (bp) LGALS3bp, T cell immunoglobulin and mucin domain 3 (TIM-3) were used. Housekeeping genes RPLP0 and UCHL5 served as internal controls. Fold changes were calculated using the Livak method. Total protein concentration was assessed using the Bicinchoninic acid assay. Secretion of GAL -1, -2, -3, -4, -8, -9 was assessed using bovine GAL specific ELISA. All tested LGALS were detected. Activation with PMA differentially modulated expression and secretion of GAL in blood and neutrophils. Expression levels of LGALS -1 and LGALS -3 was not affected. Expression of LGALS -7, -8, -9, -11, -12, and TIM-3 was differentially modulated. The secretion of GAL -3 was significantly increased in response to PMA. The most pronounced effect of PMA was observed on LGALS-9, TIM-3 expression, and the secretion of GAL -3. Thus, GAL may serve as biomarkers of cell activation with functional consequences and warrant further study.