NFAT isoforms play distinct roles in TNF alpha-induced retinal leukostasis

作者：Bretz Colin A; Savage Sara R; Capozzi Megan E; Suarez Sandra; Penn John S^*

来源：Scientific Reports, 2015, 5(1): 14963.

DOI：10.1038/srep14963

The objective of this study was to determine the role of individual NFAT isoforms in TNF alpha-induced retinal leukostasis. To this end, human retinal microvascular endothelial cells (HRMEC) transfected with siRNA targeting individual NFAT isoforms were treated with TNF alpha, and qRT-PCR was used to examine the contribution of each isoform to the TNF alpha-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 TNFa and assayed using a parallel plate flow chamber, and both NFATc2 and NFATc4 knockdown reduced TNF alpha-induced cell adhesion. The effect of isoform-specific knockdown on TNF alpha-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 TNF alpha-induced retinal leukostasis in vivo. Together, these studies show a clear role for NFAT-signaling in TNF alpha-induced retinal leukostasis, and identify NFATc2 and NFATc4 as potentially valuable therapeutic targets for treating retinopathies in which TNFa plays a pathogenic role.

• 出版日期2015-11-3