Inhibition of PTK6 Improves Intestinal Barrier Function and Claudin‐3 Expression in an Experimental Mouse Model for Ulcerative Colitis

Ulcerative colitis (UC) is a debilitating disease characterized by chronic inflammation in the gut. It is thought that increased intestinal permeability is an initial physiological mechanism that gives rise to the persistent state of inflammation. In fact, it has been demonstrated that targeting epithelial cells in a manner that increases intestinal barrier function provides patients with significant relief and fewer subsequent flare‐ups. Since protein tyrosine kinase 6 has recently been shown to play a critical role in intestinal epithelial barrier function, and PTK6 is most highly expressed in intestinal epithelial cells, we hypothesized that targeting PTK6 would improve intestinal barrier function as well as reduce clinical signs of UC. Gut mucosal barrier function was evaluated by measuring gut lumen‐to‐blood transport of a fluorescent tracer using a mouse model for experimental UC in both wild type (WT) and transgenic null mice for PTK6 expression. We found that genetic ablation of PTK6 improved gut barrier function by approximately two‐fold in the animal with experimentally induced UC. Similarly, we observed three‐fold improvement of gut barrier function in the UC mice treated with a pharmacological PTK6 inhibitor. In addition, H&E staining of colonic tissues in UC mice showed enhanced crypt structure in mice with genetic interruption of PTK6 or those treated with PTK6 inhibitor. Western blot analysis of colonic tissues showed UC mice had elevated levels of active PTK6, decreased detection of the tight junction protein claudin‐3, increased detection of pY925 focal adhesion kinase (FAK), and elevated expression of matrix metalloproteinase 9 (MMP9). Fecal supernatants from UC WT mice showed enhanced detection of cleaved E‐cadherin consistent with the size generated by MMP9 cleavage. Since PTK6 is known to phosphorylate FAK at Y925, and this phosphorylation event is important for MMP9 expression, we explored whether PTK6 interruption could enhance claudin‐3 expression and reduce the level of FAK pY925, active MMP9 and cleaved E‐cadherin in fecal supernatants. Indeed, colonic lysates from UC PTK6 null mice showed significant reductions of pY925 FAK, active MMP9 and E‐cadherin cleavage when compared to the lysates obtained from WT UC mice. Immunostaining of histological samples also showed improved tight junction organization and claudin‐3 localization at epithelial tight junctions. Taken together, these results suggest that PTK6 plays a critical role in mediating gut mucosal barrier injury in UC and may potentially regulate FAK‐dependent MMP9 activity. Support or Funding Information IBX000799, NIH HL‐120954