Identification of Integrin αvβ3 as a Receptor for the Novel Macrophage-Secreted Microprotein U9-ORF Encoded by U90926 2307000

Abstract Introduction Gene expression in macrophages is a highly dynamic and tightly regulated process to prevent inflammation-driven pathology, such as septic shock. Previous studies in our lab identified the putative long non-coding RNA (lncRNA) gene U90926 as highly induced in macrophages following pattern recognition receptor activation by lipopolysaccharide (LPS) and other microbial ligands. Using an LPS-induced sepsis model, we found that U90926 knockout mice exhibited greater disease severity and mortality, indicating a protective role of the gene. Surprisingly, analysis of U90929 sequence identified an 87 amino acid open reading frame (ORF), encoding a novel protein, U9-ORF, containing a functional N-terminal secretion signal. We therefore hypothesized that protective effects of U90926 in sepsis are mediated by the secretion of U9-ORF and binding to receptors on other cells. Methods To screen for potential receptors for U9-ORF, we developed recombinant U9-ORF-Fc fusion proteins to measure binding via flow cytometry, and for use in vitro functional assays. Transfection of HEK293 cells was used to express potential receptors for U9-ORF, and cellular adhesion assays were used as a functional readout. Results Multiple sequence alignments of predicted U9-ORF homologs revealed a conserved C-terminal RGD motif, suggesting that U9-ORF could bind specific integrins. Flow cytometry assays demonstrated that U9-ORF preferentially bound cells expressing integrin αvβ3, but not αIIbβ3, in an RGD- and divalent cation-dependent manner. Plate-bound U9-ORF-Fc supported cellular adhesion and spreading of cells expressing αvβ3 integrin, in an RGD-dependent manner. Conclusion U9-ORF is a novel ligand for integrin αvβ3, which is upregulated on endothelial cells during inflammation, and thus could mediate protective effects of U90926 in sepsis. Ongoing experiments aim to determine the effects of U9-ORF on endothelial barrier integrity in the presence of inflammatory stimuli. Funding Source NIAID/NIH R03 AI190196-01; NIAID/NIH R21 AI151116-01; Department of Education CMB-GAANN Fellowship Topic Categories Cellular Adhesion, Migration, and Inflammation (CAM)