Investigating RAGE Ectodomain Shedding (sRAGE) and Splice Variant (esRAGE) in Diabetic and Inflammatory Conditions
Authors: Ashall Louise , Thomas Sneha Ann , Inman Claire , Ramasamy Ravichandran , Schmidt Ann Marie 
Published: November 2023

Obesity, diabetes and its complications represent an increasing worldwide health epidemic, however little is known about the specific cellular and molecular mechanisms that drive these complications. The defining features of obesity and diabetes are hyperlipidemia and hyperglycemia respectively, which create a cellular environment conducive to increased formation of advanced glycation end products (AGEs). AGEs bind to its chief cell surface receptor for AGE (RAGE) thereby activating downstream signaling pathways, including NF‐κB, which leads to the transcription of genes that in part create an inflammatory environment and ultimately contribute to the development of diabetic complications.RAGE has various splice variants and isoforms, a full length (fl) membrane bound RAGE (flRAGE), which can be cleaved from the cell surface by metalloprotease (MMP9) and ADAM10 to produce soluble RAGE (sRAGE). A second form of soluble RAGE is alternatively spliced and secreted by the cell known as endogenous secretory RAGE (esRAGE or RAGEv1). Both sRAGE and esRAGE act as a decoy to RAGE as they can bind to and sequester RAGE ligands, potentially preventing ligand binding to flRAGE. The objective of this study is to investigate RAGE ectodomain shedding and the splice variant of RAGE. Although studies have examined the regulation of RAGE shedding, little investigation into both sRAGE and esRAGE in cells exposed to stresses that provoke vascular damage in diabetes, such as hyperglycemia with the addition of an inflammatory setting has yet to be explored.We hypothesized that the glucose/AGE‐stimulated upregulation of inflammatory mechanisms might contribute to the generation of soluble RAGEs. Therefore, modulation of sRAGE and esRAGE levels in cells exposed to high glucose and to the prototypic inflammatory stimulus TNFα, were measured. RAGE expressing HEK293T cells exposed to diabetes‐relevant levels of high glucose, was not sufficient to induce changes in RAGE ectodomain shedding. However high glucose together with TNFα treatment resulted in a significant (p<0.01) increase in RAGE ectodomain shedding as determined by ELISA. Western blot analysis under the same conditions showed that RAGE protein levels in the cell lysate remained unchanged indicating that the increase in ectodomain shedding was not due to increased cellular RAGE levels. The production of esRAGE remained unchanged when esRAGE expressing HEK293T cells were exposed to high glucose. Unlike RAGE ectodomain shedding, TNFα treatment alone resulted in a significant (p<0.0001) increase in RAGE splice variant levels measured by esRAGE ELISA, which was also observed at the protein level by western blot analysis. These findings were also confirmed at the endogenous mRNA level using human monocyte‐like THP‐1 cells by qPCR. Both isoforms of soluble RAGE increased in the presence of stresses that mimic diabetic complications, with the addition of TNFα treatment being a key factor. Further mechanistic studies will elucidate the regulation of increased soluble RAGE production in these conditions with the goal of identifying novel therapeutic strategies to block adverse AGE‐RAGE consequences.Support or Funding InformationNYUAD Research Institute.Al Jalila Foundation.