Introduction Keratins (K) are a key component of intermediate filaments (IF), primarily composed of K8, 18 and 19 in the intestinal epithelia. Apart from a structural role, they may play a role in moderating TNF effects, including cytotoxicity. K8-null mice develop colitis, a subset of patients with IBD have missense mutation in K8 gene. Colonic expression of K8/K18 has been shown to be regulated by IL-6. In order to examine the relationship between acute inflammation and alteration in levels of insoluble IFs in mucosa of patients with ulcerative colitis (UC), we undertook a quantitative proteomic approach using an iTRAQ (isobaric tagging for relative and absolute quantification)-based proteomic workflow.
Methods Endoscopic biopsies were obtained from patients with UC, from actively inflamed rectum and from non-inflamed proximal colonic mucosa in the same patient (n=10), and from normal controls (n=10). Endoscopic (Baron) and histological assessment was made. An iTRAQ-compatible extraction protocol for insoluble IF proteins was developed. Labelled peptides from pooled patients were analysed by SCX-LC-MS/MS (strong cation exchange-reverse phase HPLC tandem mass spectrometry) and data reconstituted in GeneBio Phenyx. Inter-group comparisons were made using in-house algorithms based on t-testing with multiple test correction.
Results Median age was 36 years (range 23–71). Endoscopic Baron score was ≥2 in inflamed mucosa for all patients. Tandem mass spectrometry (MS/MS) identified 52 proteins, 32 (61.5%) matched by two or more peptides, showing significant log fold change, with reduced levels of keratins and vimentin in inflamed mucosa compared to controls (Abstract PMO-250 table 1). Abstract PMO-250 figure 1 shows significantly reduced IF protein levels in inflamed mucosa compared to inactive mucosa. Cytokine proteins neutrophil defensin 1 and bone morphogenetic protein 4 were increased only in actively inflamed mucosa.
Conclusion Using a quantitative proteomic approach, we have shown significantly decreased levels of keratins in the actively inflamed colonic mucosa in UC providing further evidence of interaction between keratins and inflammatory pathways—which requires further elucidation.
Competing interests None declared.
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