the growth of keratinocytes in the epidermis is regulated by a delicate balance between molecules that control cell survival and cell death .
if this regulation is disturbed , epithelial cells may become pathological hyperproliferative lesions , such as cholesteatoma .
the molecular and cellular processes resulting in the clinical hallmarks of cholesteatomas ( i.e. , migration , uncoordinated proliferation , altered differentiation , and aggressiveness ) are not yet fully understood . in cholesteatoma ,
keratinocytic differentiation and proliferation may be modulated by many cytokines , transcription factors , and inflammatory mediators such as tnf- and il-1. the tumor suppressor gene cyld , which was recently identified as the cylindromatosis gene , has a deubiquitinating enzyme ( dub ) activity and inhibits activation of the transcription factor nf-b , which has key roles in inflammation , immune responses , carcinogenesis , and protection against apoptosis . in cholesteatoma
for example , nf-b was reported to be found in cholesteatoma epithelium , but it appeared to be inactivated .
however , a recent report suggested that activated nf-b plays a role in cellular hyperplasia of cholesteatoma .
generally , in normal resting cells , nf-b is localized within the cytoplasm as a heterodimeric complex composed of p50 and p65 subunits bound to members of the class of inhibitory proteins called inhibitor kappa b ( ib ) .
after various stimuli , the phosphorylation of ibs , an important step in nf-b activation , is mediated by ib kinase ( ikk ) .
the ikk complex consists of at least three subunits , including the kinases ikk- and ikk- and the regulatory subunit ikk-. phosphorylated ib is then ubiquitinated , which targets it for degradation by the 26s proteasome , thereby releasing nf-b dimers from the cytoplasmic nf-b  ib complex and allowing them to translocate to the nucleus and induce target gene expression [ 4 , 5 ] . at this point ,
cyld binds to nf-b essential modulator ( nemo)/ikk- and appears to regulate its activity through deubiquitination of tnf receptor - associated factor 2 ( traf 2 ) .
cyld was recently reported to negatively regulate nf-b signaling by deubiquitination , and loss of deubiquitinating activity of cyld is correlated with tumorigenesis [ 57 ] .
cyld also negatively regulates the c - jun n - terminal kinase ( jnk ) signaling pathway and mitogen - activated protein kinase ( mapk ) pathway , which are known to participate in a wide range of cellular processes , including proliferation , differentiation , and apoptosis of cholesteatoma [ 1 , 8 ] .
in addition , cyld negatively regulates rank signaling , which is known to be a key factor in bone resorption in cholesteatoma .
therefore , investigation of cyld expression may be a key step to understanding the cellular survival or hyperplasia in cholesteatoma epithelium .
we hypothesized that cyld may be involved in the nf-b signaling pathway and that the inverse correlation between cyld and activated nf-b level may be related to the mechanism of cellular hyperplasia in cholesteatoma epithelium . to determine the role of cyld in nf-b signaling of cholesteatoma , cyld and nf-b expression levels in middle ear cholesteatoma epithelium
were examined by immunohistochemical analysis to determine protein level and localization and compared to those of normal retroauricular ( ra ) skin .
we also examined the relationship between cyld expression and nf-b activation in middle ear cholesteatoma .
tissues of acquired cholesteatoma , diagnosed clinically and confirmed pathologically after surgery , were used in this study .
all cholesteatomas extended to the mastoid , with varying degrees of attic bone destruction with keratin retention .
the samples were obtained from patients ranging in age from 45 to 60 years ( mean age : 52 years ) who had undergone middle ear surgery for cholesteatomatous otitis media between november 2008 and may 2009 at the department of otolaryngology , kyung hee university east - west neo medical center , seoul , korea . paired normal ra skin
sixteen samples of cholesteatoma and paired normal ra skin samples were obtained for this study .
the institutional review boards ( ifc / irb ) of the kyung hee university east - west neo medical center approved the protocol used in this study ( irb approval no .
cholesteatoma and normal ra skin were fixed in 4% formaldehyde , dehydrated in a graded ethanol series ( 70% , 95% , and 100% ) , and finally embedded in paraffin .
paraffin - embedded tissues were cut into 10 m thick sections using a paraffin block cutter ( thermo shandon , runcorn , cheshire , uk ) and mounted on adhesive slides coated with poly - l - lysine ( cat .
, sections were deparaffinized , rehydrated , and treated in 10  mmol / l citric acid buffer ( ph 6.0 ) at 95c100c for 20 minutes for antigen retrieval .
endogenous peroxidase activity was blocked by incubation in 0.5% h2o2 in methanol for 20 minutes .
sections were then processed for immunohistochemistry using antibodies against cyld ( 1  :  500 dilution ; abcam inc .
, cambridge , ma ) and nf-b ( 1  :  300 dilution ; santa cruz biotechnology , santa cruz , ca ) overnight at 4c .
the abc method was used to detect cells labeled specifically using a vectastain kit ( vector laboratories , burlingame , ca ) .
the sections were subjected to a single immunostaining by enzyme - antibody method as described above . to confirm the colocalization of cyld and nf-b , two serial sections ( 1020  m thickness ) were subjected to single immunostaining using each antibodie .
images were obtained using an olympus ( tokyo , japan ) microscope and spot ( diagnostic instruments inc . ,
sterling heights , mi ) . in all immunohistochemical controls , reaction to the substrate was absent if the primary antibody was omitted or if the primary antibody was replaced by nonimmune control antibody .
three investigators blinded to the experimental groups examined immunostained sections with an image analyzer attached to a light microscope .
the total number of cells in the fixed domain , the number of cells with nuclear staining for nf-b and cyld - positive cells in the epithelial layer of cholesteatoma and normal ra skin tissues , was counted under five 400 fields in the fixed domain .
then , both the levels of activated nf-b ( number of cells with nuclear staining / total number of cells counted ) and cyld expression ( number of positively stained cells / total number of cells counted ) were obtained and expressed as percentages .      
the level of significance was set at   p < .05 . the pearson 's correlation test was used to determine the association between activated nf-b and cyld , with the level of significance set at 0.05 .
we also analyzed activated nf-b and cyld levels according to the existence of active otorrhea and state of bony destruction using the mann - whitney test .
the spss10 software package ( spss , chicago , il ) was used for the analyses .
the following results were obtained by immunohistochemical analysis of 16 cholesteatoma tissues and paired normal ra skin specimens .
immunohistochemical data indicated that immunoreactivity of cyld was mainly present in the cytoplasm and perinuclear region of the basal layer and granular layer in normal ra skin ( figure 1(a ) ) . in the cholesteatoma tissue ,
cyld expression was seen in a few cells in the basal layer ( figure 1(b ) ) .
the percentage of cyld - positive cells in cholesteatoma tissues was 38.48%  21.20% compared to 80.95%    8.08% in paired normal ra skin .
the percentage of cyld - positive cells in cholesteatoma tissue was significantly lower as compared to paired normal ra skin ( p = .022 ) .      
the differential expression of nf-b was found between normal ra skin and cholesteatoma epithelium . as shown in figure 2(a ) , nf-b expression was seen mostly in the cytoplasm ( inactivated form ) and was localized mainly in the basal epithelial layer of normal skin . in addition , we found some nuclear staining of nf-b in the basal and suprabasal layers of the normal ra skin in some cases . in cholesteatoma tissue , however , most nf-b expression was observed in the nucleus ( activated form ) and was localized in all layers of the epithelium ( figure 2(b ) ) .
the percentage of activated nf-b expression in the cholesteatoma epithelium was 62.62%  18.97% and that in paired normal ra skin was 5.47%  1.12% .
the percentage of activated nf-b expression in the cholesteatoma epithelium was significantly higher compared to normal ra skin ( p = .0001 ) .   to find the association between cyld and nf-b , we examined the correlation between cyld and activated nf-b expression .
using pearson 's correlation test , we found a significant inverse correlation between cyld and activated nf-b expression in cholesteatoma ( figure 3 , r = 0.630 , p = .007 ) .  
based on the overwhelming evidence indicating the roles of cyld and activated nf-b in immune regulation , apoptosis , and bone resorption , we examined whether the expression levels of activated nf-b or cyld are regulated according to clinical aspects , such as the presence of active otorrhea and state of bony destruction [ 6 , 9 ] .
the cholesteatoma groups in this study consisted of 6 cases with and 10 cases without active otorrhea at the time of treatment .
we found no significant differences in expression levels of nf-b or cyld between these two groups with and without active otorrhea ( p = .309 ) .       
based on operative findings , the above patients were classified retrospectively into two groups to determine the extent to which the cholesteatoma destroyed bone ; the mild group had disruption of one ossicle , while the severe group had widespread bone destruction , including disruption of more than two ossicles and destruction of the sigmoid sinus , post wall of the external auditory canal , and the facial canal .
we then compared the expression levels of cyld and activated nf-b between the two groups .
the degree of bone destruction was mild in 5 cases and severe in 11 cases in this study .
no significant relationships were observed between the degree of bone destruction and the expression levels of cyld ( p = .769 ) or activated nf-b ( p = .432 ) .
turban tumor syndrome ,  an autosomal - dominant condition that confers a predisposition to multiple skin tumors .
the gene product , cyld , is a dub recently implicated in the negative regulation of the nf-b pathway , a transcription factor suggested to promote cell proliferation [ 1 , 5 ] .
the present study was performed to examine the possible role of cyld expression in the pathogenesis of cholesteatoma .
specifically , we were interested in whether cyld expression is altered in cholesteatoma and whether cyld expression level affects the activity of nf-b in cholesteatoma .        the results of the present study indicated that the level of cyld expression was significantly downregulated in cholesteatoma in comparison with normal ra skin .
the primary function of dubs is to remove single or multiple ubiquitin chains from different proteins / substrates .
recently , different specific substrates for cyld have been identified , such as traf2/6 , nemo , and bcl-3 from which cyld could remove the ubiquitin chains by direct association [ 1 , 10 ] .
the removal of lysine63-(lys63- ) linked polyubiquitin chains from traf2 or traf6 by cyld attenuates nf-b signaling , leading to programmed cell death .
therefore , loss of the deubiquitinating activity of cyld may be correlated with tumorigenesis .       
recently , reduced cyld was also found in human carcinomas , such as hepatocellular carcinoma and colon cancer , as compared with nonneoplastic tissue .
moreover , exposure of cyld knockdown hela cells to anti - inflammatory drugs such as sodium salicylate or prostaglandin a1 , which inhibit nf-b , abolished the protective knockdown of apoptosis , suggesting that loss of cyld confers resistance to apoptosis through activation of nf-b .
as noted above , a series of reports have shown that cyld blocks signal transmission through the classical or alternative nf-b cascade by deubiquitinating the tnf receptor - associated factor 2 ( traf2 ) , traf6 , and ikk- ( nemo ) [ 1 , 5 , 6 ] .        however , a recent report showed that the loss of cyld in keratinocytes of mice did not affect transcription induced by the classical p65p50 nf-b dimers but had clear effects on bcl-3-linked p50- or p52-dependent gene regulation .
bcl-3 acts as a nuclear coactivator that switches the transcriptional properties of nf-b p50 and p52 homodimers from a repressive to an active state leading to cellular proliferation through activation of the cyclin d1 gene .
after translocation of bcl-3 from the cytoplasm to the perinuclear region , cyld removes k63-linked polyubiquitin chains from bcl-3 , and thus prevents translocation of bcl-3 into the nucleus .
these data suggest that the mechanism of cyld - mediated nf-b suppression may vary in different cell types .        in the present study
, the cytoplasmic and perinuclear expression levels of cyld suggest that cyld may play a role in the deubiquitination of bcl-3 and/or traf in nf-b signaling within the cytoplasm or perinuclear region in keratinocytes of normal skin and cholesteatoma , in agreement with previously reported results .
in addition , the present findings suggested that downregulation of cyld expression , which is highly expressed in normal skin , may be involved in the pathogenesis of human cholesteatoma .      
the role of nf-b in epithelial keratinocytes has been described as being paradoxical in other cell types such as t cells and b cells .
for example , mice overexpressing rel / p65 nf-b showed epidermal hypoplasia and growth inhibition , whereas a study on ib-null mice showed epidermal keratinocyte hyperplasia and dermal infiltration of lymphocytes .
this variation in outcome of nf-b activation may reflect the cell types and differentiation state of the cells .
lizzul suggested that the antiproliferative effects of activated nf-b in normal epithelial cells without inflammation or apoptotic stimuli may be superior to its anti apoptotic effects .      
activated nf-b , however , has an anti - apoptotic effect in proliferative skin disorders such as psoriasis . in normal murine skin ,
only basal keratinocytes can proliferate and differentiate into mature cells that undergo enucleation to generate the cornified layer .
therefore , nf-b is found in the cytoplasm ( inactivated form ) of only basal keratinocytes of normal skin .
our immunohistochemical results of normal ra skin were in agreement with these findings .        in the present study , however , significant differences in nf-b expression pattern were observed between cholesteatoma and normal ra skin . in cholesteatoma , strong immunostaining for nf-b
was seen mostly in the nucleus and was localized in not only the basal layer but also the suprabasal layers .
this was in contrast to normal ra skin , which showed nf-b immunoreactivity mostly in the cytoplasm of cells in the basal epithelial layers .
some debate exists regarding the role of nf-b in cholesteatoma [ 2 , 3 ] .
the keratinocytes in cholesteatoma are clearly in an abnormal state , and one would anticipate changes in the epidermis and keratinocyte . perhaps , this is why we observed in contrast control ra skin .
that is , cholesteatoma may represent a chronic inflammatory state , and therefore , an imbalance exists between the anti - apoptotic role and cell cycle inhibitory role of nf-b , whereby the scale is tipped toward protection against cell death in the context of a constitutive cytokine - rich inflammatory milieu .
this allows for the hyperproliferation seen in cholesteatoma .        therefore , our results suggest that activated nf-b , which may provide protection against apoptosis , plays a role in the pathogenesis of cholesteatoma , unlike normal ra skin .
vasudevan reported that the tumor suppressor pten , which functions as a negative regulator of the phosphorylated akt - mediated cell survival pathway in cholesteatoma , is downregulated by activated nf-b [ 18 , 19 ] .
these findings also support the results of the present study . to elucidate the role of cyld in the expression of activated nf-b in human cholesteatoma , we examined the correlation between cyld and activated nf-b expression in cholesteatoma .
we found a significant inverse correlation between cyld and nf-b in cholesteatoma , suggesting that nf-b activation is inhibited in cases with normal cyld function but that it tends to be overexpressed in the absence of functional cyld .
in addition , our results support the possibility that nf-b activation accompanied by loss of cyld may be an important step in the development and/or progression of cholesteatoma .
we also examined the relationships between cyld / nf-b and clinical data , such as the presence of active otorrhea and degree of bone destruction , but no such relationships were found in the present study .
in addition to the critical role of cyld in the nf-b pathway , cyld has the ability to function as a negative regulator of the jnk signaling pathway , which plays an important role in cell survival in human cholesteatoma . the precise mechanisms underlying the downregulation of cyld in cholesteatoma remain to be elucidated .
nevertheless , our data suggest the potential role of cyld inactivation in cholesteatoma epithelium via multiple mechanisms , ranging from genetic alteration to epigenetic silencing .
we found that cyld expression was lower and activated nf-b expression was higher in cholesteatoma epithelium in comparison to normal ra skin .
these observations suggest that the activation of nf-b and downregulation of cyld may be involved in the cellular hyperplasia in patients with cholesteatoma .
 the tumor suppressor cyld is a deubiquitinating enzyme that inhibits activation of the nf-b , which has key roles in inflammation and apoptosis . 
 we hypothesized that cyld may regulate the nf-b signaling pathway in cholesteatoma . 
 we conducted immunohistochemistry to examine the expression of cyld and nf-b in 16 cases of cholesteatoma and paired cases of retroauricular ( ra ) skin . in cholesteatoma epithelium , activated nf- 
 b expression was significantly higher than in ra skin , whereas cyld expression was significantly lower in cholesteatoma epithelium than in ra skin ( p < .05 ) . 
 furthermore , a significant inverse correlation was detected between cyld and activated nf-b expression in cholesteatoma epithelium ( r = 0.630 ) . 
 we found that cyld reduced and activated increased nf-b in cholesteatoma epithelium in comparison to ra skin . 
 the inverse correlation between cyld and activated nf-b in cholesteatoma may be involved in cholesteatoma epithelial hyperplasia . 
1. Introduction
2. Methods
3. Results
4. Discussion
5. Conclusions