Expression And Post-Translational Modification Of Intermediate Filament Proteins In Colonic Mucosa Of Patients With Ulcerative Colitis: Role In Pathogenesis Of Colitis Associated Colorectal Neoplasia And Dysplasia

Background
Ulcerative colitis(UC) is a chronic inflammatory disease of the colon associated with increased cancer risk of colitis associated cancer(CAC). Keratins(K) are intermediate filament(IF) proteins, and are key component of the cellular cytoskeleton. K8 null mice develop colitis; a subset of IBD patients have mutation in K8 gene. Keratins play a role in cell-death signalling pathways; epithelial cells lacking K8 and K18 are more sensitive to TNF-mediated apoptosis.
Methods
Colonic biopsies obtained from UC patients (and controls) were grouped into eight categories based on risk of cancer and presence of mucosal inflammation: quiescent recent onset (<5 years) UC (ROUC); quiescent long-standing pancolitis (20–40 years) (LSPC); UC with primary sclerosing cholangitis (PSC); active colitis (ACT) and un-inflamed proximal colonic mucosa (INACT) in the same patient; pancolitis with dysplasia-both dysplastic lesions (DT) and distal rectal mucosa (DR). An iTRAQ and western immunoblotting compatible extraction and solubilisation protocol for insoluble IF proteins was developed. Labelled peptides from pooled patient groups were analysed by quantitative proteomics. Results noted were validated by western immunoblotting.
Results
52 proteins were identified, 32(61.5%) were matched by 2 or more peptides. Acute inflammation was associated with reduced K8, K18, K19 and vimentin (p<0.05) compared to controls and un-inflamed mucosa; reduced levels were also seen in DT and DR. LSPC relative to controls or ROUC showed increased levels of IF proteins (K8, K18, K19 and vimentin, p<0.05). Multiple forms of K8 forms were identified on immunoblotting; in aggressive phenotypes relative K8 phosphorylation (K8pS23) was reduced along with an increase in vimentin:K8 ratio. Acute inflammation reduces K8 levels and phosphorylation; such changes are restored in longstanding quiescent disease LSPC but not in ROUC (despite clinical and endoscopic remission).
Conclusion
Alteration in mucosal levels of IF proteins (keratin and vimentin) may play a role in pathogenesis of colitis associated cancers.