Regulation of RAGE Splicing by iPLA2

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: MON 818-841-Diabetes Pathophysiology & Complications
Basic/Clinical
Monday, June 17, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board MON-829
Mamatha Kambalapalli*1, Xiaoyong Lei2, Sasanka Ramanadham3 and Suzanne Barbour4
1VCUHS, Richmond, VA, 2University of Alabama, Birmingham, AL, 3University of Birmingham, Birmingham, AL, 4VCU, Richmond, VA
Background: Loss of β-cell mass due to apoptosis occurs in in both type1  and type 2 diabetes mellitus. Apoptosis is regulated by a balance between proapoptotic and antiapoptotic factors. Ligation of the Receptor for Advanced Glycation Endproducts (RAGE) promotes b-cell apoptosis. RAGE undergoes alternative splicing leading to a membrane protein  (FL-RAGE- which in rodent mRNA lacks introns 9 and 10) that promotes apoptosis or a soluble protein (esRAGE- which in rodent mRNA includes introns 9 and 10) that may be protective. Group VIA phospholipase A2 (iPLA2β), a catabolic phospholipase, promotes β-cell apoptosis and our preliminary studies indicate that this is mediated at least in part through effects on pre-mRNA splicing of several pro and antiapoptotic factors.

Objective: The objective of this study is to determine if iPLA2β promotes β-cell apoptosis by regulating RAGE splicing.

Design: A chemical inhibitor (S-BEL) or siRNA was used to suppress iPLA2β in INS-1 cells. Livers were isolated from wild type and iPLA2b-/-mice. RNA was isolated, cDNA generated, and RT-PCR performed to amplify splice variants of RAGE. PCR signals were quantified and the ratio of putative full length RAGE (lacks introns 9 and 10) to putative esRAGE (contains introns 9 and 10) was determined.

Results: In INS-1 cells, iPLA2β inactivation or knockdown shifted splicing in favor of a RAGE splice variant that included introns 9 and 10, typically increasing the ratio of esRAGE/ FL-RAGE by approximately 2-fold. Similarly, iPLA2b-/- mouse liver exhibited a 2-3 fold increase in the ratio of intron 9+/ intron9- splice variant. At present, our efforts are focused on cloning and sequencing these splice variants, to confirm that they encode FL-RAGE and esRAGE. We are also using immunoblot and flow cytometry to characterize RAGE proteins in cells and tissues with high versus low levels of iPLA2b activity.

Conclusions: These data suggest that iPLA2β modulates pre-mRNA splicing of RAGE, shifting splicing to favor a variant that promotes apoptosis. A more complete understanding of the molecular mechanisms underlying iPLA2β- regulated pre-mRNA splicing may uncover novel targets to control these splicing events, suppress β-cell apoptosis, and thereby treat diabetes mellitus.

Nothing to Disclose: MK, XL, SR, SB

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm