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Expression of tyrosinated a-tubulin in Crohn's disease
AND ulcerative COLITis
Pavel Peravoshchykau 1 *, Anna Portyanko 1, Julia Gorgun 2
1 Department of Pathology, Belarussian State Medical University, Minsk, Belarus 2 Department of Gastroenterology and Nutrition, Belarusian Medical Academy of Post-Graduate Education Minsk, Belarus
ABSTRACT
Background: Tyrosinated tubulin is unique modification of a-tubulin. Its expression has been studied it number of cancer cells and was found different from normal cells. In this research, we aim on finding similar changes in epithelial cells of colonic mucosa in Crohn's disease and ulcerative colitis. We consider that it may be valuable for their diagnostics and treatment. Methods: Analysis was performed on colonic mucosa biopsy fragments: 160 pieces from patients with inflammatory bowel diseases and 52 of patients with normal colonic mucosa. Sections were stained by hematoxylin and eosin to study morphological changes in mucosa. In addition, sections were stained by double immunofluorescence method with antibodies to cytokeratin and tyrosinated tubulin. To evaluate its expression ImageJ software was used. Results: Expression of tyrosinated tubulin was found both in epithelium of inflamed and normal mucosa. This protein was highly expressed in inflammatory bowel diseases (100,5 (95%CI 94,0-107,0) vs. 84,0 (95%CI 77,7-90,3), p<0,01). Similar change of expression was found separately in Crohn's disease (133,2 (95%CI (118,6147,8) vs. 84,0 (95%CI 77,7-90,3), p<0,01). Moreover the level of tyrosinated tubulin was dependent on inflammation activity: 119,5 (95%CI 105,7-133,2) in sites of active inflammation vs. 167,9 (95%CI 132,0-203,8) in sites with non-active inflammation, (p=0,05). In ulcerative colitis the expression of tyrosinated tubulin did not significantly differ from normal control. Conclusion: In inflammatory bowel diseases expression of tyrosinated tubulin is increased in colonic mucosa epithelium. These changes are mainly seen in Crohn's disease and depend on activity of inflammation.
KEYWORDS
Inflammatory Bowel Diseases, Microtubules, Cytoskeleton, Colitis
How to cite this article: Peravoshchykau P, Portyanko A, Gorgun J. Expression of tyrosinated a-tubulin in Crohn's disease and ulcerative
colitis. Int Stud J Med. 2016; 2 (1): 25-30
INTRODUCTION
Microtubules are the main protein structures of cell cytoskeleton. They are critically important for cell shaping, mitosis, intracellular transport and secretion. Microtubules consist of aP-tubulin heterodimer. Both a- and P-tubulin subunits have numerous isoforms and posttranslational modifications, some of them are unique. In this work, we were concentrated on posttranslational modification of a-tubulin known as tyrosinated tubulin (Tyr-tubulin).
Primary form of a-tubulin after translation is mostly tyrosinated (ends on C14-tyrosine) [1, 2]. Tyr-tubulin is a predominant modification of a-tubulin in cell [3]. It is known to build labile microtubules, rich in dynamic structures such as mitotic spindle and interface network [2, 3, 4, 5]. Tyrosine can be removed by tubulin-tyrosine carboxypeptidase. Product of
Recieved: 29 February 2016/ Accepted: 13 March 2016/ Published online: 31 March 2016
* Address for Correspondence: Pavel Peravoshchykau, 220047 Ilimskaya Street, 16232, Minsk, Belarus. Tel.: +375293148097; Email: [email protected]
such reaction is called glutaminated tubulin (Glu-tubulin) - as it ends on glutamate on C-terminus. Glu-tubulin is a predominant type of a-tubulin in stable microtubules, which build axonemes, basal bodies and flagellum [2, 4, 6]. It can be retyrosinated by tubulin tyrosine ligase (TTL) [2, 4]. By these reactions, the dynamic equilibrium of Tyr- and Glu-tubulin in cell is maintained. Biochemistry of these modifications is moreless understood, but their biological role is still uncertain.
The ratio of different tubulin subtypes in microtubules of cytoskeleton is not a constant value. It was shown that microtubules remodelling is necessary for neuronal [7, 8] and muscular [9] differentiation. This process is characterised by changes of expression of different tubulin subtypes. The turnover of predominant modification of a-tubulin from tyrosinated to detyrosinated and back is particularly important for epithelial cell polarisation [10, 11].
Disruptions of tubulin remodelling was found in number of pathologic conditions. Changes of
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expression of Tyr-tubulin is best studied in cancer cells of different tissues. However, there are no studies of different tubulins in epithelial cells during inflammation.
Inflammatory bowel diseases (IBD) consist of Crohn's disease (CD) and ulcerative colitis (UC). In UC and in most cases in CD inflammation affects colonic mucosa. This chronic remitting-relapsing inflammation is believed to have an autoimmune origin. However little is known about its origin. CD and UC are much alike in morphology. This makes differential diagnosis difficult. We consider that microtubules remodeling, similar to those in cancer cells, might be a part of pathogenetic chain in these diseases. Such findings may be valuable in future for discovering new diagnostic features of CD and UC and making new effective targeting drugs.
Objective of this research: evaluation of expression of Tyr-tubulin in colonic mucosa epithelium in IBD.
METHODS
160 biopsy fragments of colonic mucosa were selected for our study. They were obtained during colonoscopy in patients with chronic colitis: 27 UC (13 female, 14 male; mean age 34,4±6,2) 12 CD (8 females, 3 males; mean age 36,2±10,3) and 1 case of undifferentiated colitis. 52 biopsy fragments were obtained from patients, to whom colonoscopy was performed as a part of differential diagnostic algorithm. 8 patients had chronic diarrhea, 3 - cases anemia and 3 -abdominal pain. IBD and other conditions that affect colonic mucosa were excluded. Following diagnoses were established: 1 case of celiac disease, 3 cases of B12-deficient anemia, 9 cases of irritable bowel syndrome, multiply sclerosis - 1 case. These patients were combined in control group (10 females, 4 males; mean age 51,8±10,0).
Biopsy samples were taken by standard diagnostic procedure - minimum 2 fragments per segment of colon. Biopsy fragments from 2 segments of colon were used in analysis. For IBD the most affected segments were chosen.
All samples were fixed in 10% neutral buffered formalin and mounted in paraffin, 4 p,m sections were made. All sections were stained by hematoxylin and eosin. Then morphologic analysis was performed. Presence and grade of inflammation were assessed.
All sections of IBD group were then divided into 2 subgroups: with active inflammation (sections with epithelial destruction or intraepithelial neutrophils) and non-active inflammation (sections with only architectural abnormalities or changes of lamina propria infiltrate).
Tyr-tubulin detection was performed by double immunofluorescence method. Primary mouse antibodies: anti-Tyr-tubulin (clone TUB-1A2, isotype IgG3, Sigma-Aldrich), anti-cytokeratin (clone AE1/3, isotype IgG1, DAKO), in 1:800 and 1:400 dilution respectively. Secondary duck antibodies: anti-IgG3 (AlexaFluor® 488) and anti-IgG1 (AlexaFluor® 555) - MolecularProbes, Invitrogen, in dilution 1:200. DAPI was used for nuclear staining. For antigen retrieval sections were heated for 2,5 min in citrate buffer solution (pH 6) in DAKO Pascal chamber. To block nonspecific binding sections were treated with 1% cow serum albumin for 30 min. Primary antibodies were incubated at 4°C overnight. For negative control staining an Isotype Control For Mouse Primary Ab was used (MolecularProbes, Invitrogen). All sections were mounted in anti-fading reagent (DAKO).
Assessment and photography of slides were performed on Leica DM5000B microscope with Leica DFC420C camera on x200 magnification. Pictures of immune-stained sections were taken at 3 fluorescent channels: A4 (blue, DAPI), L5 (green, Tyr-tubulin), Y3 (red, cytokeratin). 1 or 2 photos have been taken per section.
Images then were analyzed by program ImageJ ver.1.47t. Epithelial structures were selected on cytokeratin channel. Mask of this selection was transferred on Tyr-tubulin channel where mean fluorescence intensity was measured. All measurements were normalized to one of normal control samples repeating in every staining series.
Data were analyzed in programs STATISTICA (ver. 10.0.228.2) and Microsoft Excel 2013.
RESULTS
Tyr-tubulin was positively detected in colonic mucosa epithelium of all studied sections. It was spread equally throughout cell space. Expression of Tyr-tubulin was found significantly higher in IBD group
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Image 1. Fluorescence of Tyr-tubulin. (A) normal control, (B) IBD
Mean Confidence interval
Group Number of biopsy fragments - 95% + 95%
Control 52 84.0 77.7 90.3
IBD 160 100.5 94.0 107.0
Active inflammation 120 96.2 89.2 103.2
Non-active inflammation 40 113.3 97.9 128,6
CD 44 133.2 118.6 147.8
Active inflammation 34 119.5 105.7 133.2
Non-active inflammation 10 167.9 132.0 203.8
UC 113 88.2 81.6 94.9
Active inflammation 83 88.4 80.5 96.2
Non-active inflammation 30 90.2 79.3 101.0
Table 1. Levels of expression in studied groups
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(Image 1, Table 1, p<0,01). Moreover we found that (Table 1, Image 3A). Comparative analysis in UC
this increase is dependent on activity of inflammation failed to find any significant difference neither
(p=0,04, Table 1, Image 2A). These distinctions between UC and control group, nor between samples
happened to be statistically significant only for CD with different activity of inflammation (Image 2B,
(Image 2B). The highest level of expression was found 3B). in non-active inflammation samples of this group
Image 2. (A) Expression of Tyr-tubulin in samples with different inflammation activity (B) Expression of Tyr-tubulin in UC and CD. * - p<0,05; ** - p<0,01; *** - p<0,001.
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200
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160
140
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*** A
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r"
*** I
1
Й _
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■ 200
ISO
160
140
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100
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CD act. CD non-act.
Control
UC act.
UC non-act.
Mean X Mean±0,95 Conf. Interval
Image 3. Expression of Tyr-tubulin in samples with different inflammation activity for patients with CD (А) and UC
(B) * - р<0,05; ** - p<0,01; *** - p<0,001.
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DISCUSSION AND CONCLUSION
In present study we investigated expression of Tyr-tubulin in epithelium of normal and inflamed mucosa. There was no qualitative differences in distribution of this modification in epithelial cell. Tyr-tubulin was seen in all studied samples was spread equally alongside cell space. As predominant modification of a-tubulin, especially in interphase network, it is not expected to concentrate at cell margins. It was proved to be true: in MDCK cells Tyr-tubulin concentration was equal at basal and apical pole [11].
We found distinctions in expression of Tyr-tubulin during quantitative analysis. Level of expression was elevated in IBD. However, it was unexpected that these changes were significant only for CD. During analysis, we have detected a number of samples from UC patients where expression of Tyr-tubulin was also elevated. However, we failed to find any other similarities among them. We consider that level of Tyr-tubulin in colonic epithelial cells in UC patients also changes, but less frequently than in CD. Perhaps further analysis on this point will make it clear. It is early to state that expression of Tyr-tubulin do not change in UC.
In CD level of expression was dependent on activity of inflammation. Highest level was detected in non-active inflammation subgroup. The hallmark of inflammation is based on predominance of reparative and destructive processes in mucosa. Thus, erosions and intraepithelial neutrophils that are signs of active inflammation are morphological manifestations of destruction of epithelium. Reparation manifests with architectural abnormalities of epithelium. It is a result increased cell division rate. Therefore, there might be a connection between overexpression of Tyr-tubulin in mucosa epithelium and reparative changes in it.
Level of Tyr-tubulin in cell was found to depend on its differentiation state. Thus undifferentiated tumor cells of neuroblastoma had little of tyrosinated tubulin [7]. Artificially grown sarcomas in nude mice contained cells with suppressed TTL activity and decreased Tyr-tubulin expression [12]. These cells had selective advantage among others. Suppressed TTL activity was also seen in cells of invasive front of breast cancer [13]. However opposite results was found in colon adenocarcinoma: less differentiated cells contained more Tyr-tubulin [14].
Different tubulin modifications were studied in myogenic, fibroblastic and epithelial cell lines during differentiation [9, 10, 11]. Overexpression of Glu-tubulin was seen on early stages of differentiation before cell-cell contacts had been formed. After that in sub-confluent fibroblastic and epithelial cells and in myocytes after fusion, expression of Tyr-tubulin was seen to increase.
Thus, low expression of Tyr-tubulin in undifferentiated cancer cells may result from loss of differentiation potency. The predominant modification of a-tubulin in these cells is detyrosinated. In normal cells on late stages of differentiation expression Tyr-tubulin apparently increased.
Overexpression of Tyr-tubulin in epithelial colonic mucosa cells in IBD demonstrates their similarity to sub-confluent epithelial cells. Probably increase of expression of Tyr-tubulin in epithelium arises from increase of poorly differentiated cells in it.
Conclusion
In inflammatory bowel diseases expression of tyrosinated tubulin is increased in colonic mucosa epithelium. These changes are mainly seen in Crohn's disease and depend on activity of inflammation.
CONFLICT OF INTEREST
The authors confirm that this article content has no conflicts of interest.
AUTHOR CONTRIBUTION
All authors contributed to the study design, interpretation of the literature data, and the manuscript drafting. All authors read and approved the final version of the manuscript for publication. Research was supported by ISTC №»В1636 and ГПНИ №№1.2.42.
ORCID
Pavel Peravoshchykau http://orcid.org/0000-0002-3374-6568
Anna Portyanko http://orcid.org/0000-0003-2399-117X
Julia Gorgun http://orcid.org/0000-0002-4595-9659 REFERENCES
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ORIGINAL PAPERS
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