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DIFFERENTIAL EXPRESSION OF ZONA OCCLUDEN‐1 PROTEIN and ITS ALPHA SPLICE ISOFORMS IN CROHN'S DISEASE and ULCERATIVE COLITIS
Author(s) -
Ooi Choon Jin,
Ng Han Seong,
Podolsky Daniel K
Publication year - 2001
Publication title -
journal of gastroenterology and hepatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.214
H-Index - 130
eISSN - 1440-1746
pISSN - 0815-9319
DOI - 10.1111/j.1440-1746.2001.ca01-32.x
Subject(s) - gene isoform , tight junction , blot , alternative splicing , medicine , immunofluorescence , ulcerative colitis , cytoplasm , rock2 , microbiology and biotechnology , pathology , rna binding protein , biology , immunology , gene , messenger rna , kinase , disease , antibody , genetics , rho associated protein kinase
and aims Tight junctions (TJ) are critical in sustaining the epithelial barrier. We investigated the regulation of major TJ proteins in IBD, the MAGUK (membrane associated guanylate kinase) cytoplasmic proteins, Zona occluden (ZO)‐1 and ZO‐2. Two isoforms of TJ protein ZO‐1 exist as a result from alternative RNA splicing and they differ by an internal 80‐amino acid domain termed motif‐α. ZO‐1 a – is expressed in structurally dynamic junctions whereas ZO‐1 α + is expressed in less dynamic cells; perhaps this differentiation may correlate with the differences in junction plasticity, but the role of these different isoforms have never been studied in IBD. Methods Mucosal biopsies were obtained from patients with Crohn's disease (CD), and ulcerative colitis (UC) undergoing colonoscopies. Colonoscopic biopsies were taken from macroscopically ‘involved’ (I) and ‘not involved’ (NI) areas avoiding ulcerated areas. Control specimens were obtained from patients undergoing colonoscopies with no history or endoscopic evidence of inflammatory or infective processes. TJ proteins were evaluated by immunoblotting, and mRNAs encoding these TJ proteins were assessed by RT–PCR and Northern blotting. Cellular localization of TJ proteins was defined by immunofluorescence (IF). β‐Actin proteins were used as internal controls. A total of 40 patients were studied; 10 normal, 16 UC and 14 CD patients. Results Immunoblotting revealed down‐regulation of ZO‐1, and ZO‐2 in IBD tissues. In addition, RT–PCR showed that ZO‐1α + isoform predominated in the normal tissue while the ZO‐1α – isoform was increased in disease. Our results show that both isoforms exists in normal intestinal tissue, with the ZO‐1α – consisting 14% (SD 0.05) of total ZO‐1. However, the proportion of ZO‐1α – /total ZO‐1 increased to 29% ( P < 0.05) in uninvolved CD tissues, 40% ( P < 0.01) in inflamed CD tissues and 46% ( P < 0.001) in inflamed UC tissues. Northern blots employing DNA probes encoding both splice variants of ZO‐1 documented a decrease in steady state RNA in diseased tissues. IF showed ZO‐1 localized to dense granules on apical surfaces in normals, but was more cytoplasmic in distribution in IBD tissues. Conclusion Alteration in ZO‐1 expression may be specific changes to CD and UC. This is the first study to show up‐regulation of the ZO‐1α – in inflamed tissues in CD and UC. It is however, unclear if these changes are the primary event to leading to breakdown in barrier function or mere secondary manifestations to inflammation.