NFAT isoforms play distinct roles in TNFα-induced retinal leukostasis

• Published in: Scientific reports 2015-11, Vol.5 (1), p.14963-14963, Article 14963
• Main Authors: Bretz, Colin A., Savage, Sara R., Capozzi, Megan E., Suarez, Sandra, Penn, John S.
• Format: Article
• Language: English
• Subjects: 13; 13/106; 13/109; 38; 631/80/304; 64; 64/60; 692/699/3161/3175; 82/80; 96; Animals; Cell Adhesion - drug effects; Cell Adhesion - genetics; Cells, Cultured; Chemokine CCL2 - genetics; Chemokine CCL2 - metabolism; Chemokine CX3CL1 - genetics; Chemokine CX3CL1 - metabolism; Chemokine CXCL10 - genetics; Chemokine CXCL10 - metabolism; Chemokine CXCL11 - genetics; Chemokine CXCL11 - metabolism; Disease Models, Animal; E-Selectin - genetics; E-Selectin - metabolism; Endothelial Cells - metabolism; Enzyme-Linked Immunosorbent Assay; Gene Expression; Humanities and Social Sciences; Humans; Intercellular Adhesion Molecule-1 - genetics; Intercellular Adhesion Molecule-1 - metabolism; Leukostasis - chemically induced; Leukostasis - genetics; Leukostasis - metabolism; Male; Mice, Inbred C57BL; multidisciplinary; NFATC Transcription Factors - genetics; NFATC Transcription Factors - metabolism; Protein Isoforms - genetics; Protein Isoforms - metabolism; Retinal Diseases - chemically induced; Retinal Diseases - genetics; Retinal Diseases - metabolism; Retinal Vessels - cytology; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Science; Tumor Necrosis Factor-alpha - pharmacology; Tumor Necrosis Factor-alpha - toxicity; Vascular Cell Adhesion Molecule-1 - genetics; Vascular Cell Adhesion Molecule-1 - metabolism
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/srep14963

The objective of this study was to determine the role of individual NFAT isoforms in TNFα-induced retinal leukostasis. To this end, human retinal microvascular endothelial cells (HRMEC) transfected with siRNA targeting individual NFAT isoforms were treated with TNFα and qRT-PCR was used to examine the contribution of each isoform to the TNFα-induced upregulation of leukocyte adhesion proteins. This showed that NFATc1 siRNA increased ICAM1 expression, NFATc2 siRNA reduced CX3CL1 , VCAM1 , SELE and ICAM1 expression, NFATc3 siRNA increased CX3CL1 and SELE expression and NFATc4 siRNA reduced SELE expression. Transfected HRMEC monolayers were also treated with TNFα and assayed using a parallel plate flow chamber and both NFATc2 and NFATc4 knockdown reduced TNFα-induced cell adhesion. The effect of isoform-specific knockdown on TNFα-induced cytokine production was also measured using protein ELISAs and conditioned cell culture medium and showed that NFATc4 siRNA reduced CXCL10, CXCL11 and MCP-1 protein levels. Lastly, the CN/NFAT-signaling inhibitor INCA-6 was shown to reduce TNFα-induced retinal leukostasis in vivo . Together, these studies show a clear role for NFAT-signaling in TNFα-induced retinal leukostasis and identify NFATc2 and NFATc4 as potentially valuable therapeutic targets for treating retinopathies in which TNFα plays a pathogenic role.