Intestinal cell barrier function in vitro is severely compromised by keratin 8 and 18 mutations identified in patients with inflammatory bowel disease.

Tina Zupancic,Jure Stojan,Radovan Komel,Apolonija Bedina-Zavec,Mirjana Liovic,Ellen Birgitte Lane,Markus M Heimesaat

doi:10.1371/journal.pone.0099398

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.

Highlights

In the last 20 years intermediate filaments (IF) have been recognized as an important component of the cytoskeleton filament system and to have a variety of functions, far beyond the initial simplistic view of a mechanical support to cells
Clones of HT-29 colonocytes stably transfected with K8 and K18 wild type and mutant constructs (K8 WT, K8 G62C, K8 K464N, K18 WT and K18 S230T) were engineered and selected according to the methodology described in Materials and Methods
By introducing the K8 (G62C and K464N) and K18 (S230T) mutations into the background of the same wild type HT-29 colonocyte cell line, we created isogenic cell lines and eliminated potential variations arising from different genetic backgrounds

Summary

Introduction

In the last 20 years intermediate filaments (IF) have been recognized as an important component of the cytoskeleton filament system and to have a variety of functions, far beyond the initial simplistic view of a mechanical support to cells. IF proteins are being used in diagnostics as markers of distinct physiological changes in tissues during tumorigenesis [9], [10]. The amazing progress made on IF protein biology started in the early 90’s, when the first keratin gene mutations were identified and linked to a group of skin blistering disorders [11], [12]. Keratins are the largest family of IF proteins and are specific to epithelial tissues. A number of mutations have been identified in the simple epithelia keratins K8, K18 and K19 [13]. These are associated with pathologies of the liver, pancreatitis and inflammatory bowel disease (IBD). On the contrary to epidermal keratins, which when mutated mostly have a dominant-negative effect, the simple epithelia keratins appear to act as a susceptibility factor

Objectives

Methods

Results

Conclusion