Investigating the Role of Cdc42 in the Production and Release of Pro-Inflammatory Mediators in Airway Epithelial Cells
Creators: Shouib, Rowayna
Publisher: name: University of Alberta Library
Year: 2024

<p>Epithelial cells line the lung airways providing a protective physical and immunological barrier against inhaled particles and pathogens. Hence, airway epithelial cells express a variety of surface receptors that confer recognition of inhaled toxins, allergens and various pathogens. Receptor engagement is coupled with release of pro-inflammatory molecules that recruit and activate immune cells and orchestrate inflammatory responses at the lungs. While this release is essential for host defense responses against infections, it can exacerbate existing inflammation in the lungs, especially in chronic conditions of asthma and COPD patients. Since the regulation of cytokine gene expression and secretion from epithelial cells is not fully characterized, we sought to investigate whether this process is regulated by Rho GTPase proteins. Rho GTPases act as molecular switches and their downstream signaling mediators control a variety of inflammatory pathways. Specifically, we focus on the GTPase Cdc42 which is implicated in inflammatory gene expression and is known to have a Golgi pool, suggesting a potential role in cytokine trafficking.<br>
In Chapter 3, we investigate the role of Cdc42 in bronchial epithelial cell inflammation by ablating its activity to induce a loss of function. This is achieved through the use of the pharmacological inhibitor, ML141, and through genetic silencing mediated by shRNA knockdown. We report that inhibition of Cdc42 with ML141 or shRNA-mediated silencing differentially modulates pro- inflammatory cytokine mRNA levels and as well as their secretion. IL-8 levels increase while MCP-1 levels decrease after ML141 treatment. These effects are concomitant with a disruption of cytokine trafficking patterns through the secretory pathway. In addition, we report a disruption to Golgi structure and integrity. Cdc42 inhibition or knockdown result in an aberrant fragmented phenotype.<br>
In Chapter 4, we report on a transcriptome analysis by RNA sequencing to investigate the genome-wide changes occurring at the mRNA level upon inhibition of Cdc42 with ML141. Our analysis reveals a gene enrichment in inflammatory pathways and transcriptional regulators, as well as in ER stress pathways due to ML141 treatment. In addition, we identify differentially expressed signaling proteins that might have a role in mediating these effects. We show that these targets, specifically TRIB3 and DUSP5, are downstream effectors of Cdc42 that couple inflammatory gene expression to Cdc42 inhibition. We also show other targets, namely SESN2 and BMP4, to be involved in maintaining Golgi integrity downstream of Cdc42 as their genetic depletion blocks ML141-induced Golgi fragmentation. We also characterize perturbations to gene sets linked to specific compartments within the Golgi and to different stages along the secretory pathway for changes in gene expression.<br>
In Chapter 5, we investigate the effects of Cdc42 mutants with alternative lipidations that result in aberrant localization to subcellular compartments, such as the Golgi and the nucleus. We investigate whether an enrichment at these compartments is associated with trafficking defects or enhancements in order to understand the importance of the different Cdc42 pools in the cell. We do not find significant changes to cytokine trafficking or Golgi integrity. This is likely due to the normal activity of endogenous Cdc42 that obscures the effects of exclusive sequestration in different organelles.<br>
Taken together, our findings reveal a role for Cdc42 as a dual regulator of cytokine dynamics. It can both positively and negatively regulate cytokine production while being a positive regulator of trafficking. In addition, we also interrogate the cellular inflammation network induced by Cdc42 inactivation, characterizing genetically enriched pathways and identifying signaling targets that mediate inflammatory properties.</p>