The DNA Binding Capacity of RAGE and its Consequences in the Development of Diabetic Nephropathy — ASN Events

The DNA Binding Capacity of RAGE and its Consequences in the Development of Diabetic Nephropathy (#94)

Brooke Harcourt 1 , Aaron McClelland 2 , Hiroshi Yamamoto 3 , Angelika Bierhaus 4 , Phillip Kantharidis 2 , Josephine M Forbes 1
  1. Mater Medical Research Institute, South Brisbane, QLD, Australia
  2. Diabetes Complications, Baker IDI Heart and Diabetes Institute, Melbourne, VIC, Australia
  3. Department of Biochemistry and Molecular Vascular Biology, Kanazawa University, Kanazawa, Japan
  4. Department of Internal Medicine I and Clinical Chemistry, University of Heidelberg, Heidelberg, Germany

Background/Aim: Multiple isoforms of the receptor for advanced glycation end products (RAGE) have previously been identified. These include a full-length membrane bound isoform (fl-RAGE), soluble isoforms (sRAGE and esRAGE) and a membrane sequestered N-truncated isoform (N-RAGE). The same gene encodes all of these isoforms. We wanted to investigate if RAGE can bind within its own promoter region ultimately acting as a transcription regulator for its own expression.
Methods: Eight to ten week old male high fat fed C57BL/6J mice with genetic deletions of receptor for advanced glycation end products (RAGE-/-, C57BL/6JRAGE-/-) or leptin receptor, db/db or db/H (db/db, Lepr (+/+)C57BL/KsJ, db/H, Lepr (+/-)C57BL/KsJ) were followed for 16 or 10 weeks respectively. Chromatin was immunoprecipitated from renal cortices, using antibodies to known RAGE transcription factors, NF-ĸB, Sp-1, and estrogen receptor (ER). Chromatin was also immunoprecipitated for RAGE. The genomic DNA was then analysed for transcriptional activity in the 5’ transcriptional binding domain of the RAGE gene using EMSA and RT-PCR to identify specific consensus binding sites. The expression of RAGE in the nucleus was further confirmed by ELISA and immunohistochemistry in renal cortex sections. NRK cells were analysed for the transcription of RAGE by using transiently transfected luciferase reporter plasmids containing specific regions of the transcriptional domain of RAGE, These were compared to TNF-α treatment a known inducer of RAGE expression.
Principle Findings: In conditions of worsening renal disease the expression of nuclear RAGE in renal cortices increases. Furthermore, the DNA transcriptional regulation of the RAGE gene via RAGE binding in its promoter region was associated with NF-ĸB in these renal tissues. We also identified the specific DNA binding sequence where RAGE biding in its own promoter occurs which was confirmed by EMSA and supershift. RAGE as a self-transducible gene regulator was further confirmed in our cell culture experiments whereby luciferase activity significantly increased in TNF-α/RAGE induced experimental groups.

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