PSVI-4 Modulation of galectin gene expression and secretion in cow blood stimulated with Phorbol 12-myristate 13-acetate

Abstract

Abstract The objective of this study was to assess the expression and secretion of galectin gene in cow blood stimulated with Phorbol 12-myristate 13-acetate (PMA). Phorbol 12-myristate 13-acetate is a phorbol diester that activates the enzyme protein kinase C (PKC). Activated PKC is associated with regulation of inflammation. Galectins (Gal) are β-galactoside binding proteins that modulate inflammation following intra and extra-cellular secretion. Blood was collected from the jugular vein of clinically healthy Holstein-Friesian cows (n = 3) from North Carolina A&T diary unit. Individual samples were treated with either 10ng/ml of PMA or Phosphate buffer saline (PBS) for 30 minutes at 370 C. Total RNA was isolated from blood, pooled and transcribed to cDNA, for real-time PCR. Cow specific primers LGALS -1, -2, -3, -4, -7, -8, -9, -12 were designed using the NCBI Primer 3 tool. Housekeeping genes RPLP0 and UCHL5 were used for normalization. Fold change was calculated using the Livak method (Fold change >2 were considered as significant). Secretion of GAL -1, -2, -3, -4, -8, -9 was assessed using bovine GAL specific ELISA kits. Data were analyzed by one-way ANOVA using SAS version 9.4. Treatment with PMA up regulated the transcription of LGALS -2, -4, -8, (2.28, 2.36, 2.39 folds respectively) and down regulated the transcription of LGALS -9 (-5.04 fold). Transcription of LGALS -1, -3, -7, 12 remained unchanged. All tested GAL were secreted. Treatment with PMA increased the secretion level of GAL -1, -3, -4 and decreased the secretion level of GAL -2, -4, -9 (P > 0.05). Thus, activation of PKC differentially modulates galectin gene expression and secretion in cow blood. The observed differences in treatment and control groups warrant further studies.