O-GlcNAcylation Regulates Angiotensin II-dependent Induction of Thrombospondin 1

O-Linked attachment of β-N-acetylglucosamine (O-GlcNAc) on serine and threonine residues of nuclear, cytoplasmic, and mitochondrial proteins is a highly dynamic and ubiquitous post-translational modification that plays an important role in altering the function, activity, subcellular localization, and stability of target proteins. It has been proposed that physiologically, O-GlcNAcylation serves primarily to modulate cellular signaling and transcription regulatory pathways in response to nutrients and stress. However, in the vascular physiology literature, O-GlcNAc has predominantly been shown to be detrimental. Our preliminary data has shown that O-GlcNAcylation can cause aortic stiffness, however, the mechanisms underlying this phenotype remain to be elucidated. The predominant mechanisms by which O-GlcNAcylation contributes to alterations in proteostasis include competition with other post-translational modifications as well as regulation of gene expression. Thrombospondin-1 (TSP1) is a multifunctional matrix glycoprotein that impacts extracellular function (ECM) and cell adhesion, migration, proliferation, and cell-cell interaction. Therefore, we hypothesized that TSP1 could be an important downstream mediator of O-GlcNAcylation in aortic vascular smooth muscle cells (VSMCs). To test this hypothesis, primary VSMCs from the thoracic aorta were isolated from 12-week-old female Wistar rats and submitted to angiotensin (ANG) II treatment (100 nM, 3h). Some cells were also treated in the presence of ANG II type 1 (AT1)-receptor antagonist losartan (10−5 M, 30 min). ANG II was chosen because it is a physiological stressor known to cause arterial stiffening. O-GlcNAcylation (RL2 antibody), O-GlcNAcase (OGA), and TSP1 protein amounts were analyzed by Western blot, and the results were expressed in arbitrary units (AU) and mean ±standard error mean. Unpaired Student's test T was used and p<0.05 was considered statistically significant. Genetic and pharmacological inhibition of O-GlcNAc was achieved through OGT silencing and inhibition with a shRNA (FunGene6) and OSMI-4 (10−5 M, 30 min), respectively. As hypothesized, ANG II significantly increased O-GlcNAc in aortic VSMC (0.3±0.03 vs 0.5±0.03 AU n=8, p<0.05) which was inhibited by losartan (0.1±0.02). Moreover, ANG II significantly decreased OGA expression (0.16±0.04 vs 1.16±0.19 AU n=4, p<0.05), the enzyme responsible for removing O-GlcNAc motifs, supporting the observed increase in O-GlcNAc expression. Importantly, ANG II also increased TSP1 protein expression (0.2±0.04 vs 2.0±0.4 n=4, p<0.05), however, shRNA (0.7±0.05) for OGT and OSMI-4 (0.06±0.08) prevented this increase. Overall, these data indicate that Ang II-AT1R signaling can cause O-GlcNAcylation and that TSP1 could be an important downstream mediator of O-GlcNAc-mediated aortic stiffness. National Institutes of Health (R00HL151889, P20GM109091-Pilot Project, and P20GM103641-Pilot Project). University of South Carolina Offce of Research ASPIRE Award (180950-23-64136). American Heart Association Postdoctoral Fellow (916031). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.