Introduction Intermediate filaments (IF) are one of the main components of the human cell cytoskeleton with keratins (K) being the largest component. K8, K18 and K19 constitute the main keratins in the intestinal epithelial cells. Keratin alteration may play a role in the pathophysiology of ulcerative colitis (UC). K8 -/- mice develop chronic colitis. K8 and K18 play a role in TNF-α induced-apoptosis. We have previously shown increased expression of insoluble K8, K18 and K19 in long-standing UC relative to recent – onset ulcerative colitis (≤5 years) using mass spectrometry (MS) in the IF fraction of pooled patient samples. The aim of this study was to use antibody-based relative quantification of K8, K18, K19 in individual patient samples to validate MS results and describe variation in expression across the cohort.
Methods IF proteins were extracted from individual biopsies in patients with long-standing pan-colitis (LSPC) in clinical, endoscopic and histological remission (n = 10) and with recent onset ulcerative colitis (ROUC) also in remission (n = 8). Each sample was dot-blotted on a membrane followed by immunoblotting for identification and quantification of keratins (8, 18 and19) sequentially. MCF-7 cell line was used as control in each experiment. Relative Keratins concentration for each dot- blotted sample was inferred by determining its signal intensity relative to the MCF-7 keratins signal intensity measured in turn by densitometry. Statistical analysis to compare the two groups was made separately for K8, K18, K19 using Mann-Whitney U test.
Results Median relative IF protein levels in patients with LSPC were 1.54, 0.41 and 2.12 for K8, K18 and K19, respectively were significantly higher than those with ROUC: 0.03, 0.05 and 0.07 for K8 (p = 0.001), K18 (p = 0.002) and K19 (p = 0.021), respectively. Median Baron’s endoscopy score in patients with LSPC and ROUC were 0 (range 0–1) and 1 (range 0–1), respectively. Median histological activity index in both groups were 0 (range 0–1).
Conclusion This study confirms increased expression of insoluble keratins in colonic epithelial cells during LSPC in remission relative to the levels in ROUC and validate our previous MS observations. Restoration of keratins in quiescent LSPC may be a protective mechanism against recurrent inflammation and colorectal cancer.
Disclosure of Interest None Declared.
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