AM ER IC AN JOUR NA L OF OTOLARY NG OLOG Y –H EA D A N D N E CK ME D I CI NE AN D SUR G E RY 3 5 ( 2 0 14 ) 13 0–1 3 6
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Jak-Stat signaling pathway may play a role in the pathogenesis of cholesteatoma☆,☆☆,★ Görkem Eskiizmir, MD a,⁎, H. Seda Vatansever, MD b , Erdogan Özgür, MD a , Asım Aslan, MD a , Gökçe Tanyeri, MD a , Derya Gözüaçık, PhD a , M. Kemal Özbilgin, MD b , Cemal Cingi, MD c a b c
Department of Otolaryngology-Head & Neck Surgery, Celal Bayar University, Manisa, Turkey Department of Histology & Embryology, Celal Bayar University, Manisa, Turkey Department of Otolaryngology-Head & Neck Surgery Osmangazi University, Eskişehir, Turkey
ARTI CLE I NFO
A BS TRACT
Article history:
Purpose: Jak-Stat signaling pathway is one of the major signal transduction cascades which
Received 10 July 2013
regulates most of the cellular events such as cell proliferation, differentiation, cell migration and apoptosis. This study aims to determine the activity of Jak-Stat signaling pathway in the pathogenesis of cholesteatoma. Materials and Methods: Cholesteatoma and skin samples were obtained from 10 patients who underwent tympanomastoidectomy for chronic otitis media with cholesteatoma. Immunohistochemical analysis of cholesteatoma and skin was performed using anti-Jak1, anti-Jak2, anti-Jak3, anti-Stat1, anti-Stat2, anti-Stat3, anti-Stat4 and anti-Stat5 antibodies. The immunoreactivities in cholesteatoma and skin were quantified using H-score measurement and statistical comparison was performed. Results: Jak1, Jak2, Jak3, Stat1 and Stat3 immunoreactivities were not detected in cholesteatoma; in contrast to the skin (129.8; 226.7; 33.0; 66.4;115.9). In addition, when Hscore measurements of Stat2, Stat4 and Stat5 immunoreactivities were compared between cholesteatoma (172.8; 166.7; 120.0) and skin (400.0; 284.9; 292.0), statistically significant differences were found (p < 0.0001, p < 0.0001, p < 0.0001). Conclusions: A remarkable deficiency in the family members of Jak-Stat signaling pathway was demonstrated in cholesteatoma. Therefore, perturbations in Jak-Stat signaling pathway may play a role in the pathogenesis of cholesteatoma. © 2014 Elsevier Inc. All rights reserved.
1.
Introduction
Cholesteatoma is a chronic disease that is characterized by migration of keratinized hyperproliferative squamous epithelium located in the temporal bone [1]. The epithelium of cholesteatoma is known as the matrix, and loose epithelial
connective tissue including collagen fibers, fibroblasts and inflammatory cells is also known as perimatrix. Histologically, the epithelium of cholesteatoma is similar to the epidermis of skin; however, uncontrolled keratinocyte hyperproliferation is one of the major differences between skin and cholesteatoma. In a histopathological examination of cholesteatoma, epithelial
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Financial Disclosure: None. Conflict of Interest: None. ★ This study was presented in the 9th International Conference on Cholesteatoma and Ear Surgery; June 3–7, 2012; Nagasaki-Japan ⁎ Corresponding author at: Department of Otolaryngology-Head Neck Surgery, Celal Bayar University, 45010, MANİSA-TURKEY. Tel. +90.535.327 85 52; fax: + 90.236.237 02 13. E-mail address:
[email protected] (G. Eskiizmir). ☆☆
0196-0709/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.amjoto.2013.10.005
AM ER IC AN JOUR NA L OF OTOLARY NG OLOG Y –H EA D A N D N E CK ME D I CI N E AN D SUR G E RY 3 5 ( 2 0 14 ) 13 0–1 3 6
atrophy (78%), epithelial acanthosis (88%), hyperplasia of the basal layer (88%) and formation of epithelial cones (62%); all of which indicate the hyperproliferative behavior of cholesteatoma, were reported [2]. In addition, Kim et al. investigated the role of hyperproliferative and migratory processes of keratinocytes in the pathogenesis of cholesteatoma in an experimental model [3]. They observed an increase in cytokeratin expressions as the cholesteatoma formed, and significant changes in CK13/ 16 expression, an important indicator for hyperproliferation, were detected. Therefore, they concluded that uncontrolled keratinocyte hyperproliferation has an important role in the pathogenesis of cholesteatoma. In literature, the relationship between cytokines and uncontrolled keratinocytes hyperproliferation in cholesteatoma has been reported in several studies [4–9]. Cytokines are low density proteins in structure and constitute an important part in innate and adaptive immunity. They also play a key role in immune reponse and inflammation by providing intercellular communications. The biological functions of cytokines such as cell activation, proliferation, differentiation, and survival or death are triggered by signal transduction cascades. One of the major cellular signalling pathways for cytokines is Janus tyrosine kinase (Jak)/signal transducers and activators of transcription terminators (Stat) signaling pathway. Cytokines are connected to the cell surface cytokine receptors (types I and II), cause dimerisation and activate
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receptor Jak tyrosine kinases. Activated Jaks create “binding sites” on the latent form of Stats by phosporylated specific tyrosine residuals. Phosphorylated Stats enter the nucleus where they can bind specific regulatory sequences to activate or repress transcription of target genes (Fig. 1). Therefore, an optimal Jak/Stat activity is the major determinant for normal transmission of a cytokine or growth factor signal [10]. Current investigations have revealed that there are four members of Jak family of proteins (Jak1, Jak2, Jak3 and Tyk2). There is a selectivity between Janus protein kinase proteins and cytokines; Jak1 (interleukin-2, 4, 6, 7, 9, 10, 11, 15, 21, 22, interferon-α/β and γ), Jak2 (interleukin-3, 5, 12, 23, leptin, prolactin and interferon-γ), Jak3 (interleukin-2, 4, 7, 9, 10, 11, 15, and 21), Tyk2 (interleukin-12, 23 and interferon-α/β) [11,12]. Stat family involves six structurally and functionally related proteins: Stat1 (interferons), Stat2 (interferon-α/β and interferon- γs), Stat3 (IL-6, IL-10 and interferon -α/β), Stat4 (IL12 and IL-23 and interferon -α), Stat5 (growth hormones, prolactin, epidermal growth factor, IL-3, granulocyte/macrophage colony stimulating factor, erythropoetin, IL-2, IL-4, IL-5, IL-7, IL-9 and IL-15), Stat6 (IL-4, IL-13 and interferon-α) [13,14]. It has been known that dysregulation of Jak/Stat signalling pathway may cause immunodeficiencies and cancers [15,16]. In addition, any perturbation in Jak/Stat signaling pathway may predispose dysregulation of cellular proliferation, differentiation, and apoptosis. As a matter of fact, all of these
Fig. 1 – The Jak/Stat signaling pathway. Cytokines connect to the cell surface cytokine receptors and cause dimerisation and activate receptor Jak tyrosine kinases. Activated Jaks create "binding sites" on the latent form of Stats by phosphorylated specific tyrosine residuals. Phosphorylated Stats enter the nucleus where they can bind specific regulatory sequences to activate or repress transcription of target genes.
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Table 1 – The value and and statistical comparison of Jak and Stat immunoreactivities in cholesteatoma and skin.
Jak1 cholesteatoma Jak1 skin Jak2 cholesteatoma Jak2 skin Jak3 cholesteatoma Jak3 skin Stat1 cholesteatoma Stat1 skin Stat2 cholesteatoma Stat2 skin Stat3 cholesteatoma Stat3 skin Stat4 cholesteatoma Stat4 skin Stat5 cholesteatoma Stat5 skin
Case 1
Case 2
Case 3
Case 4
Case 5
Case 6
Case 7
Case 8
Case 9
Case 10
Mean
0 192.0 0 285.0 0 18.0 0 62.0 180.0 400.0 0 85.0 200.0 285.0 176.0 360.0
0 180.0 0 294.0 0 44.0 0 80.0 174.0 400.0 0 132.0 200.0 300.0 80.0 285.0
0 40.0 0 175.0 0 36.0 0 60.0 196.0 400.0 0 76.0 120.0 294.0 95.0 200.0
0 184.0 0 160.0 0 34.0 0 76.0 186.0 390.0 0 184.0 150.0 225.0 120.0 370.0
0 145.0 0 300.0 0 26.0 0 68.0 166.0 420.0 0 110.0 143.0 315.0 154.0 220.0
0 130.0 0 225.0 0 28.0 0 84.0 178.0 370.0 0 80.0 134.0 304.0 75.0 286.0
0 75.0 0 210.0 0 32.0 0 56.0 186.0 430.0 0 96.0 110.0 276.0 165.0 308.0
0 108.0 0 190.0 0 48.0 0 70.0 140.0 410.0 0 78.0 220.0 260.0 90.0 300.0
0 164.0 0 178.0 0 50.0 0 60.0 154.0 380.0 0 198.0 210.0 290.0 184.0 340.0
0 80.0 0 250.0 0 14.0 0 48.0 168.0 400.0 0 120.0 180.0 300.0 67.0 260.0
0 129.8 0 226.7 0 33.0 0 66.4 172.8 400.0 0 115.9 166.7 284.9 120.0 292.0
processes have a significant role in the pathogenesis of cholesteatoma. Hence, we considered that examining the potential role of Jak/Stat signalling pathway in the pathogenesis of cholesteatoma was worth to investigate.
2.
Patients and methods
This study protocol was approved by the Ethical Committee of Osmangazi University. Skin and cholesteatoma specimens were obtained from 10 patients who underwent tympanomastoidectomy for chronic otitis media with cholesteatoma (n = 10). All surgeries were performed by the same surgeon (A. A.). Skin specimens were obtained from external acoustic meatus after surgical incision. Both skin and cholesteatoma specimens were collected for histopathological and immunohistochemical examination.
2.1. Histopathological and immunohistochemical examination The sections were stained using indirect immunohistochemistry method for the immunohistochemical analyses. The sections were incubated at 60 °C overnight and then dewaxed in xylene for 30 min. After soaking in a decreasing series of
p value 0.0005 0.0005 0.0005 0.0005