Intestinal Cell Barrier Function In Vitro Is Severely Compromised by Keratin 8 and 18 Mutations Identified in Patients with Inflammatory Bowel Disease

Abstract

Keratin 8 and 18 (K8/K18) mutations have been implicated in the aetiology of certain pathogenic processes of the liver and pancreas. While some K8 mutations (K8 G62C, K8 K464N) are also presumed susceptibility factors for inflammatory bowel disease (IBD), the only K18 mutation (K18 S230T) discovered so far in an IBD patient is thought to be a polymorphism. The aim of our study was to demonstrate that these mutations might also directly affect intestinal cell barrier function. Cell monolayers of genetically engineered human colonocytes expressing these mutations were tested for permeability, growth rate and resistance to heat-stress. We also calculated the change in dissociation constant (Kd, measure of affinity) each of these mutations introduces into the keratin protein, and present the first model of a keratin dimer L12 region with in silico clues to how the K18 S230T mutation may affect keratin function. Physiologically, these mutations cause up to 30% increase in paracellular permeability in vitro. Heat-stress induces little keratin clumping but instead cell monolayers peel off the surface suggesting a problem with cell junctions. K18 S230T has pronounced pathological effects in vitro marked by high Kd, low growth rate and increased permeability. The latter may be due to the altered distribution of tight junction components claudin-4 and ZO-1. This is the first time intestinal cells have been suggested also functionally impaired by K8/K18 mutations. Although an in vitro colonocyte model system does not completely mimic the epithelial lining of the intestine, nevertheless the data suggest that K8/K18 mutations may be also able to produce a phenotype in vivo.

A novel function for the MAP kinase SMA-5 in intestinal tube stability

“…Interestingly, keratin polymorphisms have been associated with intestinal bowel disease in human patients (Owens and Lane, 2004; Owens et al. , 2004; Zupancic et al. , 2014).…”

Multiple roles for keratin intermediate filaments in the regulation of epithelial barrier function and apico-basal polarity

“…Unfortunately, detailed studies of intestinal permeability at various levels are missing. However, intestinal cells that express K8 or 18 bearing mutations identified in patients with Inflammatory Bowel Disease (IBD), showed an impaired barrier function, suggesting an epithelial cell-autonomous mechanism by which TJ permeability is dependent on IF 98 . Therefore, increased barrier permeability is a possible mechanism linking deficient keratin expression (or disease associated keratin mutations) and inflammation.…”

Potential Regulatory Effects of Corticotropin-Releasing Factor on Tight Junction-Related Intestinal Epithelial Permeability are Partially Mediated by CK8 Upregulation

“…Zupancic et al first reported that CK8 mutations could cause dysfunction of the intestinal epithelial barrier and abnormal distribution of TJ proteins Claudin-4 and ZO-1 [13]. CK8 overexpression has been shown to induce degradation and remodelling of actin and tubulin [14-17]; its negative correlation with TJ-related proteins has also been reported [18].…”