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Jing Lu, Chen Yang, Colin J Barnstable, Samuel Shaomin Zhang; Specific Expression of Tnfaip3/A20 by An Alternative Transcriptional Start-Site in Mouse Retina. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4504.
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© ARVO (1962-2015); The Authors (2016-present)
Tnfaip3/A20 is required for terminating NFkappaB signaling in response to microbial products such as lipopolysaccharide (LPS). Loss of Tnfaip3/A20 functions has been associated with many autoimmune diseases in human. We have reported that Tnfaip3/A20 has an alternative transcriptional start-site specifically in mouse retina, however the functions of Tnfaip3/A20 in retina remain unclear. In this study we explored characterizations of Tnfaip3/A20 in response to LPS in retina compared to the response in spleen of same animals.
Animal use was in accordance with ARVO and IACUC guidelines at Penn State University College of Medicine. C57BL/6j mice (Jackson Lab) in experimental groups were treated intraperitoneally with LPS from Escherichia coli 055:B5 (Sigma-Aldrich). Saline was used as control. Retina and spleen tissues were collected from each animal. RNA extraction/purification (Qiagen).and RNA reverse transcription (Invitrogen) followed the manufacturer’s protocol. Two sets of primers were designed specifically to distinguish the RNA products from two transcriptional start-sites. Beta-actin was served as internal RNA control. PCR reaction was employed to examine the products. For quantitative analysis, the intensity of RT-PCR band was analyzed via ImageJ (version 1.46r) and further statistical analysis was done by unpaired Student t-test or one-way ANOVA based on the experimental designs. The expression of Tnfaip3/A20 during mouse retina development was analyzed from a previous dataset generated from this lab.
The developmental expression pattern of Tnfaip3/A20 matched perfectly that of a set of well-known retina-specific genes including Rs1h, Abca4 and Rho, but differed from the expressions of Tnfaip1 or Tnfaip2, other members of the same gene family as Tnfaip3/A20. In response to LPS treatment, the level of Tnfaip3/A20 from spleen extracts presented a de novo expression and reached the peak at 1 hour after LPS treatment, which agreed with many previous studies. However the expression of Tnfaip3/A20 from retina tissues showed a higher level at the beginning, decreasing at 1 hour and reached the lowest level at 24 hours.
A distinct responding pattern to LPS treatment between retinas and spleens was uncovered in this study. Giving the important role of Tnfaip3/A20 in NFkappaB pathway, further investigation of Tnfaip3/A20 functions in retina is needed.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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