Human thymic epithelial cell: untransfected control vs. scramble-siRNA transfected vs. CD49e-siRNA transfected

BACKGROUND: The thymus is a central lymphoid organ, in which bone marrow-derived T cell precursors undergo a complex process of maturation. Developing thymocytes interact with thymic microenvironment in a defined spatial order. A component of thymic microenvironment the thymic epithelial cells (TEC) are crucial for the maturation of T-lymphocytes because the secretion of extracellular matrix compounds, thymic hormones and cytokines and also by cell-cell contacts which can be mediated by extracellular matrix compounds and its receptors, integrins. There is evidence that extracellular matrix molecules play a fundamental role in localizing the different thymocyte stages in the thymus. The interaction between alpha5beta1 (CD49e/CD29; VLA-5) and fibronectin is essential for thymocyte adhesion. Previous results from our laboratory have shown that adhesion of thymocytes to cultured TEC line is enhanced in the presence of fibronectin. And we also demonstrated that anti-VLA-5 antibody when applied to growing TEC prior to the co-culturing with thymocytes, significantly prevented their adhesion to the epithelial. RESULTS: Herein, we studied the role of integrin alpha5 subunit in thymocyte and thymic epithelial cell interactions. We investigate the in vitro effects of silencing alpha5 integrin subunit in human thymic epithelial cell line by RNA interference. After silence a great deal of interest has arisen on intracellular signaling due the absence of alpha5 integrin subunit silence. The expression profile using cDNA microarrays of alpha5 subunit silenced cells showed 131 genes differential expression. Our data showed downregulation in two genes with cell adhesion function: neuronal cell adhesion molecule (NrCAM) and Activin receptor-like kinase 1 gene and in genes related with integrin pathway like, Phosphatidylinositol-4-phosphate 5-kinase, Phospholipase C gamma1 and SHC (Src homology 2 domain containing) transforming protein 1. We also assessed the effect of the loss of alpha5beta1 expression on TEC-thymocytes interaction and ours experiments confirm that alpha5beta1 contribute to adhesion rate. CONCLUSION: Conjointly, our data reinforce the importance of VLA-5 in TEC thymocyte interactions in the process of thymocyte differentiation and we hypothesize a decrease in stress fibers due absence of alpha5 integrin subunit and a compensatory pathway because of downregulation of neuropilin-2, vascular endothelial growth factor and plexin A. Three-condition experiment, control TEC vs. Scramble-siRNA TEC vs. CD49e-siRNA TEC. Biological replicates: 3 control, 3 Scramble-siRNA TEC and 3 CD49e-siRNA TEC transfected, independently grown and harvested. Two replicate per array.