Abstract
Purpose
Jak – Stat signaling pathway is one of the major signal transduction cascades which 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- Jak 1, anti- Jak 2, anti- Jak 3, anti- Stat 1, anti- Stat 2, anti- Stat 3, anti- Stat 4 and anti- Stat 5 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 H-score measurements of Stat 2, Stat 4 and Stat 5 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.
1
Introduction
Cholesteatoma is a chronic disease that is characterized by migration of keratinized hyperproliferative squamous epithelium located in the temporal bone . 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 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 . In addition, Kim et al. investigated the role of hyperproliferative and migratory processes of keratinocytes in the pathogenesis of cholesteatoma in an experimental model . 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 . 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 receptor Jak tyrosine kinases. Activated Jak s create “binding sites” on the latent form of Stat s by phosporylated specific tyrosine residuals. Phosphorylated Stat s 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 .
Current investigations have revealed that there are four members of Jak family of proteins ( Jak 1, Jak 2, Jak 3 and Tyk2). There is a selectivity between Janus protein kinase proteins and cytokines; Jak 1 (interleukin-2, 4, 6, 7, 9, 10, 11, 15, 21, 22, interferon-α/β and γ), Jak 2 (interleukin-3, 5, 12, 23, leptin, prolactin and interferon-γ), Jak 3 (interleukin-2, 4, 7, 9, 10, 11, 15, and 21), Tyk2 (interleukin-12, 23 and interferon-α/β) . Stat family involves six structurally and functionally related proteins: Stat 1 (interferons), Stat 2 (interferon-α/β and interferon- γs), Stat 3 (IL-6, IL-10 and interferon -α/β), Stat 4 (IL-12 and IL-23 and interferon -α), Stat 5 (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), Stat 6 (IL-4, IL-13 and interferon-α) .
It has been known that dysregulation of Jak / Stat signalling pathway may cause immunodeficiencies and cancers . 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 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 ethanol, sections were washed with distilled water. They were then treated with 2% trypsin in 50 mM Tris buffer (pH 7.5) at 37 °C for 15 min, and washed with phosphate-buffered saline (PBS). Sections were delineated with an Elite Pap pen (DBS, Pleasanton, CA, USA) and incubated in 3% H 2 O 2 (K31355100, Merck, Darmstadt, Germany) for 5 min at room temperature to inhibit endogenous peroxidase activity. They were washed three times for 5 min with PBS. Then sections were incubated with blocking serum (ready to use according to manufacturer’s instructions: 85-9043, Invitrogen, Camarillo, CA, USA) for 1 h. Primary antibodies: Jak 1, Jak 2, Jak 3, Stat 1, Stat 2, Stat 3, Stat 4 and Stat 5 in a dilution of 1:100 were applied at 4 °C for overnight. Next, the sections were incubated with biotinylated IgG (supplied ready to use) for 30 min, followed by three washes of PBS and then with streptavidin–peroxidase conjugate (supplied ready to use) for 30 min (85-9043, Invitrogen, Camarillo, CA, USA) and washed with PBS three times. The sections were then incubated with a solution containing diaminobenzidine (DAB, 00-2020, Invitrogen, Camarillo, CA, USA) 50 μL for each section for 5 min to visualize immunolabeling. After rinsing with distilled water, slides were counterstained with Mayer’s hematoxylin (02274390059, J.T.Barker, Deventer, The Netherlands) for 5 min. They were then washed with distilled water again, they were dehydrated with 80% and 95% alcohol and immersed in xylene and covered with mounting media (H701, CC/Mount, Universal Phosphatase Kit, Diagnostic Bio- Systems, Pleasanton, CA, USA). The negative controls received the same treatment as described above, but were incubated with rabbit IgG or mouse IgG instead of the primary antibodies. They were then evaluated under a light microscope (Olympus BX40, Olympus Corp., Tokyo, Japan).
The intensity of immunostaining for each parameter ( Jak 1, Jak 2, Jak 3, Stat 1, Stat 2, Stat 3, Stat 4, and Stat 5) was graded as: (i) no staining: 0, (ii) weakly stained: 1, (iii) moderately stained: 2 and (iv) strongly stained: 3. In addition, H – score measurement [Σn (i + 1); n: percentage of immunostained cells, i: intensity of immunostained cells] was performed for cholesteatoma and skin specimens individually.
2.2
Statistical analysis
All the results were evaluated using SPSS ver. 10.0 for Windows (SPSS, Chicago, IL). H – score values of cholestatoma and skin were entered into an SPSS file for statistical comparison. The statistical significance was analyzed by Mann–Whitney U test and p values < 0.05 were considered statistically significant.
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 ethanol, sections were washed with distilled water. They were then treated with 2% trypsin in 50 mM Tris buffer (pH 7.5) at 37 °C for 15 min, and washed with phosphate-buffered saline (PBS). Sections were delineated with an Elite Pap pen (DBS, Pleasanton, CA, USA) and incubated in 3% H 2 O 2 (K31355100, Merck, Darmstadt, Germany) for 5 min at room temperature to inhibit endogenous peroxidase activity. They were washed three times for 5 min with PBS. Then sections were incubated with blocking serum (ready to use according to manufacturer’s instructions: 85-9043, Invitrogen, Camarillo, CA, USA) for 1 h. Primary antibodies: Jak 1, Jak 2, Jak 3, Stat 1, Stat 2, Stat 3, Stat 4 and Stat 5 in a dilution of 1:100 were applied at 4 °C for overnight. Next, the sections were incubated with biotinylated IgG (supplied ready to use) for 30 min, followed by three washes of PBS and then with streptavidin–peroxidase conjugate (supplied ready to use) for 30 min (85-9043, Invitrogen, Camarillo, CA, USA) and washed with PBS three times. The sections were then incubated with a solution containing diaminobenzidine (DAB, 00-2020, Invitrogen, Camarillo, CA, USA) 50 μL for each section for 5 min to visualize immunolabeling. After rinsing with distilled water, slides were counterstained with Mayer’s hematoxylin (02274390059, J.T.Barker, Deventer, The Netherlands) for 5 min. They were then washed with distilled water again, they were dehydrated with 80% and 95% alcohol and immersed in xylene and covered with mounting media (H701, CC/Mount, Universal Phosphatase Kit, Diagnostic Bio- Systems, Pleasanton, CA, USA). The negative controls received the same treatment as described above, but were incubated with rabbit IgG or mouse IgG instead of the primary antibodies. They were then evaluated under a light microscope (Olympus BX40, Olympus Corp., Tokyo, Japan).
The intensity of immunostaining for each parameter ( Jak 1, Jak 2, Jak 3, Stat 1, Stat 2, Stat 3, Stat 4, and Stat 5) was graded as: (i) no staining: 0, (ii) weakly stained: 1, (iii) moderately stained: 2 and (iv) strongly stained: 3. In addition, H – score measurement [Σn (i + 1); n: percentage of immunostained cells, i: intensity of immunostained cells] was performed for cholesteatoma and skin specimens individually.
2.2
Statistical analysis
All the results were evaluated using SPSS ver. 10.0 for Windows (SPSS, Chicago, IL). H – score values of cholestatoma and skin were entered into an SPSS file for statistical comparison. The statistical significance was analyzed by Mann–Whitney U test and p values < 0.05 were considered statistically significant.
3
Results
All the results including H-score values for all cases, mean and p values were represented in Table 1 . In cholesteatoma, Jak 1, Jak 2, Jak 3, Stat 1 and Stat 3 immunoreactivities were absent; on the other hand, Jak 1, Jak 2, Jak 3, Stat 1 and Stat 3 immunoreactivities were detected in skin specimens ( Figs. 2, 3, 4, 5 and 7 ). In addition, the comparison of cholesteatoma and skin demonstrated a statistically significant difference for Jak 1, Jak 2, Jak 3, Stat 1 and Stat 3. Stat 2, Stat 4 and Stat 5 immunoreactivities were detected in both cholesteatoma (mean values of 172.8, 166.7 and 120.0) and skin (mean values of 400.0, 284.9 and 292.0) specimens ( Figs. 6, 8 and 9 ). However, the differences between the H-scores of cholesteatoma and skin were statistically significant for Stat 1, Stat 4 and Stat 5.
