Post-Transcriptional Regulation of Integrin ß4E and Its Role in Cell Physiology

Acute lung injury (ALI) and ventilator-induced lung injury (VILI) are severe clinical syndromes with high mortalities. They are characterized by pulmonary endothelium dysfunction, including cytoskeleton rearrangement, barrier integrity loss, and protein-rich fluid leakage into the lung, leading to compromised gas exchange. Endothelial focal adhesions (FAs), collections of proteins including transmembrane integrins which anchor the cell to the extracellular matrix (ECM), are central to organizing the cytoskeleton and propagate cellular signals. One such integrin, integrin β4 (ITGβ4) has been shown to play an active role in ALI and VILI. Although ITGβ4 binds to the ECM extracellularly, which strengthens the endothelial barrier and protects against ALI and VILI, signaling via its intracellular domain is responsible for proinflammatory effects which favors ALI/VILI development. ITGβ4 has five splice variants, termed A-E. ITGβ4E is unique in its much shorter intracellular portion than the other variants, and thus may only exhibit a barrier protective phenotype. We sought to understand how ITGβ4E is produced and its physiological role. In chapter 1, we review ITGβ4’s place in the focal adhesion of endothelial cells, and its possible role in endothelial barrier regulation during ALI/VILI. In chapter 2, we explore how ITGβ4E is produced, especially how splicing of ITGβ4E is regulated. We found that simvastatin, a barrier protective agent, upregulates ITGβ4, especially E. Two barrier compromising agents, TNFα and LPS, downregulated ITGβ4, especially E. A splicing factor, SRSF1, was found that partially controls splicing. Two other proteins, SRSF9 and SAMD4A, directly or indirectly control ITGβ4 mRNA levels. SAMD4A was found to be regulated by simvastatin, TNFα, and LPS, and thus likely is responsible for the effects of those agents. Seven single nucleotide polymorphisms (SNPs) were found that also influence ITGβ4 splicing. In chapter 3, we evaluate the functional role of ITGβ4E in endothelial cells. We proved that ITGβ4E exhibits protection against thrombin-induced endothelial barrier integrity loss. We then looked at the effects of SAMD4A, and found that removing SAMD4A enhances endothelial barrier strength, this enhancement is dependent on ITGβ4, but this does not affect the damage done by thrombin. In these experiments, we also discovered that thrombin induces ITGβ4 cleavage at a site likely not found in ITGβ4E. We also evaluated some of the changes elicited by simvastatin. We found that simvastatin treatment caused ITGβ4 to distribute on the basal and lateral sides of endothelial cells, and to cluster at sites of cell-cell junctions. ITGβ4E on the other hand only exhibited a basal distribution. We verified that ITGβ4E, like the longer variants, heterodimerizes with ITGα6. In chapter 4, we turn to the role of ITGβ4E in cancer. Multiple mutations found in cancerous tissue samples were predicted to influence ITGβ4 splicing. Using cultured esophageal squamous cells, the type of cell where one of these mutations was found, we observed that ITGβ4E slows cell migration while the longer variants enhance migration. We also found that many splicing changes are found in cancer tissues compared to their healthy tissue counterparts. Overall, this shows that ITGβ4E is regulated by many factors and has a distinct role from other variants in endothelial and cancer physiology