The vascular endothelium's principal responsibility is to support the inflammatory and metabolic needs of each of the body's organ systems. As such, these cells are highly sensitive to environmental cues and must be able to respond in a precise manner. The regulation of these responses likely occurs via numerous competing pathways. Of particular importance are the fluid shear stress forces imparted by blood flow. The mechanosensitive transcription factors KLF2 and KLF4 compete with other mechanosensitive transcription factors such as NF-kB and SMAD2/3 to suppress inflammation and vessel remodeling. Defects in the coupling between these competing pathways can cause vascular malformations. Klf2/4 are transcriptionally regulated by a MAPK-complex consisting of the kinases MEKK2/3, MEK5, and ERK5, and the scaffold protein p62. Importantly, the p62-MAPK-Klf2/4 axis is activated by high shear but suppressed by low shear to permit low shear-induced inflammation and remodeling. Besides the MAPKs, p62 also interacts with mitochondrial proteins and polyubiquitinated proteins. As mitochondrial remodeling and proteotoxic stress responses have been implicated in both shear responses and p62 signaling, this project will explore the possibility that shear-dependent changes to mitochondrial or ubiquitin homeostasis acts as a key regulator of p62-MAPK signaling. In aim 1 we will test shear-dependent changes to mitochondrial function and the role of mitochondria in regulating p62-MAPK-Klf2/4 signaling. In aim 2 we characterize shear-dependent changes to the ubiquitinome, determine its role in regulating p62-MAPK-Klf2/4 signaling, and the role of ubiquitin homeostasis regulating crosstalk between mechanotransduction pathways. The results obtained in this project will provide insight into the regulation of homeostatic mechanotransduction pathways and may reveal genetic and environmental drivers of KLF2-4-associated pathologies.