IRF6 is Required for Cell‐Extracellular Matrix Adhesions

Abstract

Patients with mutations in Interferon Regulatory Factor 6 (IRF6) display increased post-surgical wound healing complications. IRF6 is a transcription factor that functions as a master regulator of keratinocyte differentiation.. We demonstrated that IRF6 is also necessary for keratinocyte migration. Cellular adhesions are essential structures for cell migration. They mediate attachments between cells to the extracellular matrix (ECM), or between cells themselves, and these two different types of adhesions are known to regulate each other. Keratinocytes deficient for IRF6 show defective subcellular localization of components of cell-cell adhesions. Hence, we hypothesized that IRF6 also regulates cell-ECM adhesions, specifically focal adhesions. To test our hypothesis, we took advantage of human keratinocyte cell lines knocked down for IRF6 (shIRF6) and evaluated their ability to attach to plastic or Collagen IV. We found that shIRF6 keratinocytes show a delay attaching to Collagen IV compared to wild-type cells. However, no differences were observed on plastic. We further evaluated the ability of these cell lines to attach to the ECM by exposing keratinocytes in culture to centripetal forces and quantify their detachment from the plates. We found higher detachment of shIRF6 keratinocytes on Collagen IV but no differences on plastic. Plastic is stiffer than Collagen IV, hence, we hypothesized that the stiffness of the ECM would affect how IRF6 mediates cell-ECM adhesions. Using polyacrylamide gels of different stiffness, we found that shIRF6 keratinocytes have a significant reduction in their migrational speed on soft gels (1.5 kPa) compared to wild-types. Reduction of IRF6 also caused more deformation of the ECM. To test if increased cellular contractility mediated by the actomyosin cytoskeleton was driving the observed deformation, we performed immunostaining for Myosin IIb and filamentous actin. We found no visible differences in filamentous actin. However, Myosin IIb was significantly increased in shIRF6 compared with wild types, both on soft and stiff ECMs. Cell-ECM attachments are mediated by focal adhesion protein complexes including vinculin and beta1-integrin amongst others. We found that both were significantly reduced in shIRF6 keratinocytes regardless of the stiffness of the ECM. Overall, our data demonstrate that IRF6 is required for cell-ECM adhesion, potentially by regulating the localization of focal adhesion components at the cell membrane. Furthermore, IRF6 may promote cell-ECM adhesions differently depending on the stiffness of the ECM. As wounded tissue is stiffer than healthy tissue, these results are clinically significant and could contribute to the understanding of wound healing defects in patients with IRF6 mutations.