title: NFAT isoforms play distinct roles in TNFα-induced retinal leukostasis.
abstract: 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.
authors.affiliation: Vanderbilt University Medical Center, Department of Cell and Developmental Biology, Nashville, TN, USA.
authors.forename: Colin A
authors.initials: CA
authors.lastname: Bretz
authors.affiliation: Vanderbilt University Medical Center, Department of Pharmacology, Nashville, TN, USA.
authors.forename: Sara R
authors.initials: SR
authors.lastname: Savage
authors.affiliation: Vanderbilt University Medical Center, Department of Molecular Physiology and Biophysics, Nashville, TN, USA.
authors.forename: Megan E
authors.initials: ME
authors.lastname: Capozzi
authors.affiliation: Vanderbilt University Medical Center, Department of Cell and Developmental Biology, Nashville, TN, USA.
authors.forename: Sandra
authors.initials: S
authors.lastname: Suarez
authors.affiliation: Vanderbilt University Medical Center, Department of Cell and Developmental Biology, Nashville, TN, USA.|Vanderbilt University Medical Center, Department of Pharmacology, Nashville, TN, USA.|Vanderbilt University Medical Center, Department of Molecular Physiology and Biophysics, Nashville, TN, USA.|Vanderbilt University Medical Center, Department of Ophthalmology and Visual Sciences, Nashville, TN, USA.
authors.forename: John S
authors.initials: JS
authors.lastname: Penn
mesh_terms: D000818:Animals; D002448:Cell Adhesion / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000235:genetics / Q000378:metabolism; D054428:Chemokine CX3CL1 / Q000235:genetics / Q000378:metabolism; D054357:Chemokine CXCL10 / Q000235:genetics / Q000378:metabolism; D054371:Chemokine CXCL11 / Q000235:genetics / Q000378:metabolism; D004195:Disease Models, Animal; D019040:E-Selectin / Q000235:genetics / Q000378:metabolism; D042783:Endothelial Cells / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics / Q000378:metabolism; D018921:Leukostasis / Q000139:chemically induced / Q000235:genetics* / Q000378:metabolism; D008297:Male; D008810:Mice, Inbred C57BL; D050778:NFATC Transcription Factors / Q000235:genetics* / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D012164:Retinal Diseases / Q000139:chemically induced / Q000235:genetics* / Q000378:metabolism; D012171:Retinal Vessels / Q000166:cytology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology* / Q000633:toxicity; D019010:Vascular Cell Adhesion Molecule-1 / Q000235:genetics / Q000378:metabolism
chemical_list: C111835:CCL2 protein, human; C515538:CX3CL1 protein, human; C114753:CXCL10 protein, human; C515202:CXCL11 protein, human; D018932:Chemokine CCL2; D054428:Chemokine CX3CL1; D054357:Chemokine CXCL10; D054371:Chemokine CXCL11; D019040:E-Selectin; C573420:ICAM1 protein, human; D050778:NFATC Transcription Factors; D020033:Protein Isoforms; C546936:SELE protein, human; D014409:Tumor Necrosis Factor-alpha; D019010:Vascular Cell Adhesion Molecule-1; D018799:Intercellular Adhesion Molecule-1