tumor necrosis factor receptor - associated factors ( trafs ) act as adapter molecules controlling signaling pathways , such as nuclear factor kappa b ( nf-b ) , interleukin-1 receptor ( il-1r ) , toll - like receptor ( tlr ) , and transforming growth factor- ( tgf- ) [ 1 , 2 ] . for a long time
it is important that traf6 contributes to the cd40-mediated activation of nf-b and c - jun kinase ( jnk ) .
association of traf6 with cd40 is essential for cd40-mediated il-6 expression , which could explain the requirement for membrane - bound cd40 ligand to induce il-6 production by immunocytes . the survival , regulation , and activation of immunocyte and epithelial cell , signaling through cell surface receptors to activate nf-b and mitogen - activated protein kinases ( mapks ) through traf6 , are critical regulations of immune response .
unlike traf6 , the molecular mechanism of traf4 in multiple signaling pathways triggered by tnfr - related proteins remains enigmatic .
it has been indicated that traf4 augments nf-b activation through glucocorticoid - induced tnfr ( gitr ) expression on t cells , b cells , and macrophages . as a unique traf family member mediating signal transduction by tnf , il-1r , or tlr , it is found that traf4 acts as a positive effector of bone morphogenetic protein ( bmp ) and the tgf- signaling pathway .  
the intestinal epithelium and immune cells in the gut establish active sites of immune reactivity .
breakdown of homeostasis between intestinal microbiota and the mucosal immune system , together with both environmental and genetic factors , leads to inflammatory bowel disease ( ibd ) .
nf-b and tlr are considered as nodal points in the suppression and/or recruitment of immune responses in ibd .
interestingly , although mechanisms of trafs in ibd are not yet fully studied , traf - related inflammatory mediators as tgf- or cd40 play critical roles in a wide array of cellular functions in ibd [ 9 , 10 ] .   recent studies in immune cells and transgenic mice regarding the role of traf4 and traf6 have revealed that they share the same binding sites , yet comprehensive study of traf4 and traf6 in ibd is still lacking . based on the hypothesis that traf4 and traf6 may be activated prior to the clinical or endoscopic activation in patients with ibd , we sought to measure traf4 and traf6 expressions to explore their potential roles in ibd patients .
patients with pregnancy , colorectal resection for uc , disease involving only small bowel , poor bowel preparation as visible area of intestinal mucosa < 90% , or use of steroids , immunosuppressants , or infliximab before colonoscopic sampling were excluded . no diagnosis altered after at least 3 months of followup .
endoscopic score was evaluated using simplified endoscopic score in crohn 's disease ( ses - cd ) or baron score for patients with cd or uc , respectively .
human intestinal biopsies and blood samples were collected at division of gastroenterology and hepatology , shanghai jiao - tong university school of renji hospital medicine , in accordance , with guidelines of the research ethics committee of renji hospital , shanghai jiao tong university , school of medicine .
human tissue specimens were taken from both macroscopically inflamed and non - inflamed regions of the colon .
tissue specimens were put into liquid nitrogen within 10 minutes after biopsy for protein extraction or kept in rnalater ( qiagen ) for rna isolation .
human peripheral blood was separated into plasma and peripheral blood mononuclear cells ( pbmcs ) .
plasma was obtained using commercially edta - treated tubes ( gongdong medical technology co. , ltd . ) and pbmcs were isolated according to lymphoprep ( axis - shield poc as , norway ) protocol .
briefly , diluted blood was overlayed over 3  ml lymphoprep and centrifuged at 800  g for 20  mins .
then , pbmcs were kept in rnalater ( qiagen ) for rna isolation according to manufacturer 's protocol .
plasma was obtained following centrifugation of whole blood for 15 minutes at 2,000  g .
samples were analyzed using kits against traf4 and traf6 , according to the manufacturer 's specifications ( lanji biochemical and diagnostics , shanghai , china ) and a microtiter plate reader was used to read absorbance at 450  nm .
total rna in pbmcs and tissue samples were isolated with trizol reagent ( ambion ) according to the manufacturer 's protocol for cells and tissue .
the quantity and quality of rna were detected using a nanodrop 1000 ( nanodrop technologies , wilmington , de , usa ) .
primers were designed with primer 5.0 ( abi ) software and consequently synthesized by sango biotech ( shanghai ) co. , ltd .
the primer set for traf4 was 5-aggagttcgtctttgacaccatc-3 ( forward ) and 5-ctttgaatgggcagagcacc-3 ( reverse ) , with a product of 162  bps .
the primer set for traf6 was 5-cctttggcaaatgtcatctgtg-3 ( forward ) and 5-ctctgcatcttttcatggcaac-3 ( reverse ) , with a product of 140  bps .
the primer set of gapdh was 5-gtgaaggtcggagtcaacgg-3 ( forward ) and 5-cctggaagatggtgatgggat-3 ( reverse ) , which provided a product of 226  bps .
cdnas were produced with primescripttm rt reagent kit ( takara biotechnology dalian co. , ltd . ) .
briefly , reverse transcripts were incubated at 37c for 15 minutes and 85c for 5 seconds .
sybr premix ex taq kit was purchased from takara and real - time pcr reactions were done using a stepone plus device ( applied biosystems ) at 95c for 10 seconds followed by 40 cycles of 95c for 5 seconds and 60c for 20 seconds according to instruction of the sybr premix ex taq kit .
the expression levels of the target genes were normalized to gapdh with 2-ct method . for western blot analysis , pmbcs and tissue samples
were lysed in ripa buffer ( sigma ) containing protease inhibitors ( roche ) and agitated on ice for 30 minutes .
protein concentrations were determined using pierce bca protein assay kit ( pierce , wohlen , switzerland ) .
protein electrophoresis was performed according to the protocol of mini - protea iii ( bio - rad ) .
briefly , proteins were separated in 10% polyacrylamide gels ( tris / glycine ) and transferred onto polyvinylidene fluoride membrane ( millipore ) .
western blots were probed with antibodies against traf4 ( rabbit polyclonal anti - traf4 ; 1  :  1000 , santa cruz ) , traf6 ( mouse monoclonal anti - traf6 , 1  :  1000 , santa cruz ) , and -actin ( mouse monoclonal anti--actin ; 1  :  2500 , santa cruz ) .
statistical significance was determined using graphpad prism 5.0 for windows ( graphpad software , san diego , ca , usa ) .
a p < 0.05 was considered significant with either anova analysis or tukey 's multicomparison .
a two - sided fisher 's exact test or  test was performed to analyze discrete variables .
from february 2007 to february 2010 , 40 cd patients , 42 uc patients , and 40 healthy controls were included in our present study .
patients with ibd indicated significantly lower body mass index ( bmi ) than healthy controls ( p < 0.0001 ) .
the uc group contained significantly more smokers than cd patients ( 22  :  8 , p = 0.0023 ) .
three patients with ileitis , 15 patients with ileocolitis , and 22 patients with colitis were enrolled in cd group .
thirteen patients with proctosigmoiditis , 20 patients with left sided colitis , and 9 patients with pancolitis were enrolled in uc group .
characteristics of included subjects were described in table 1 . to investigate the diagnostic value of traf4 and traf6 in ibd , we detected levels of soluble traf4 and traf6 in plasma of ibd patients .
we found that traf4 and traf6 were significantly higher both in patients with cd and uc than in healthy controls ( figures 1(a ) and 1(b ) ) .
however , only overexpression of soluble traf4 showed a significantly positive correlation with endoscopic disease activity index ( baron score ) in uc patients ( spearman 's r = 0.458 , p = 0.002 ) .
furthermore , we observed that traf4 showed a significantly diagnostic value in differentiating active ibd patients from healthy controls ( p < 0.0001 , figures 2(a ) and 2(b ) ) .
although traf6 also showed a significantly diagnostic value in differentiating active cd , uc from healthy controls , the lower area under the curve ( auc ) predicted a less diagnostic value than traf4 ( figures 2(c ) and 2(d ) ) .   to identify gene expressions of traf4 and traf6 in pbmcs in patients with cd and uc , we isolated rna from pbmcs .
similar to their expression in plasma , traf4 and traf6 showed significantly higher levels both in patients with cd and uc than in healthy controls ( figures 1(c ) and 1(d ) ) ( all p < 0.0001 ) . given that segmental changes can exhibit inflammation in the colon in ibd patients and that intestinal segments in endoscopic remission can appear as histologic colitis , we determined the expressions of traf4 and traf6 both in inflamed and non - inflamed intestinal mucosae . unfortunately , 3 patients with only ileitis and nine patients with pancolitis were excluded based on exclusion criteria .
quantitative real - time pcr ( qrt - pcr ) was used to determine the gene expressions of traf4 and traf6 in inflamed and non - inflamed intestinal mucosae of ibd .
it was found that traf4 and traf6 expressions were significantly higher in inflamed intestinal mucosa of patients compared to normal mucosa of healthy controls ( all p < 0.0001 ) ( figures 3(a ) and 3(b ) ) .
interestingly , traf6 expressions were also significantly higher in non - inflamed tissue of ibd patients than in healthy controls , which may indicate potential preactivation of traf6 in ibd ( figure 3(b ) ) .
western blotting was used to measure protein expressions of traf4 and traf6 in inflamed and non - inflamed intestinal mucosae of ibd ( figure 4 ) .
our data indicate that only traf6 expressions were significantly higher in non - inflamed tissue of ibd patients than in healthy controls , although traf4 and traf6 protein expressions were significantly higher in inflamed intestinal mucosa of patients than in normal mucosa of healthy controls ( all p < 0.0001 ) ( figures 3(c ) and 3(d ) ) .
similar to their gene expressions , traf4 and traf6 protein expressions were significantly increased in inflamed intestinal mucosa compare to the non - inflamed mucosa or healthy controls .
also , traf6 protein expression was significantly higher in inflamed intestinal mucosa of ibd patients compared to healthy controls .
in the current study , we demonstrated that two members of the traf family , traf4 and traf6 , were activated in patients with ibd .
although both traf4 and traf6 showed potentially diagnostic value in differentiating active cd and uc from healthy controls , only traf6 could be pre - activated in non - inflamed tissue of ibd patients .
although trafs have similar overall structural features including a c - terminal receptor - binding domain and a leucine - zipper domain , their own structural difference leads to distinctive interaction with receptors [ 13 , 14 ] .
growing evidence indicates that trafs are regulated not only by their own structural features but also by the nature of their interactions , recruitment , or localization .
early overexpression studies clearly indicated that traf6 contributes to the cd40-mediated activation of nf-b and other signaling molecules .
nf-b activity has been upregulated in lamina propria immune cells and in epithelia of the inflamed gut in ibd .
the nf-b pathway regulates inflammation , regulatory t - cell production , and dc function .
however , activation of important transcriptional regulators including nf-b and the stress - activated protein kinases ( sapks ) mediated by traf6 requires binding to cd40 .
the cytoplasmic cd40 binding domain for traf6 is necessary for the cd40-mediated activation of il-6 production in monocytes and macrophages , and the proinflammatory il6 biologic network is upregulated in active ibd .
moreover , experiments with immune cells or fibroblasts isolated from traf6-deficient mice indicate that traf6 is required for the cd40-mediated activation of not only nf-b but also jnk and p38 mapk signals [ 19 , 20 ] .
although cd40-mediated jnk activation in b cells seems to require cytoplasmic traf6 , traf6 mutants defective in cd40 binding were able to activate the jnk pathway and upregulate cd80 , indicating that traf6 may be able to contribute to certain jnk signals without the binding of cd40 .
jnk and mapk signaling pathways are involved in governing lymphocyte influx into the gut in ibd patients by regulating lymphocyte adhesion and transmigration .
furthermore , only one study screened potential alterations of the traf6 gene in a large number of cd and uc patients but failed to identify apparent disease - causing mutations .
their data suggested that traf6 may have essential roles in human biology that it might not tolerate any significant structural alterations .
dendritic cells from traf4-deficient mice exhibited reduced migration both in vitro and in vivo experiments .
this result indicates that traf4 could participate in immune functions by facilitating immune cell migration .
interestingly , traf4 increases nf-b activation through the gitr via a traf - binding site located in the cytoplasmic domain of gitr .
this domain is responsible for the inhibition of treg cells and the promotion of t - cell activation .
although no nf-b activity has ever been detected via traf4 overexpression , traf4 has been implicated as an upstream molecule that regulates the jnk pathway via interaction and activation of misshapen ( msn ) , a member of the sps1 protein kinase family . moreover
, traf4 positively regulates transforming growth factor ( tgf)- via potentiating bone morphogenetic protein ( bmp ) and nodal signaling .
accordingly , traf4 is upregulated in b cells following cd40 signaling , which suggests that traf4 affected downstream of cd40 pathway .
this may explain our result that traf4 expressions were not significantly different in non - inflamed tissue of ibd patients compared to healthy controls .  
our understanding of traf4 and traf6 functions in the present study indicates that overlapping and unique functions will ultimately be attributed to each of the trafs . to our knowledge
, this paper may be the first one to combine analyzing traf4 and traf6 in ibd patients .
first , the present study did not include histological criteria to distinguish non - inflamed tissue from inflamed tissue , which may confuse the microscopically inflamed tissues with non - inflamed tissues .
second , the present study could not include laboratory data as esr or crp to assess activity of disease , which may lead to the potential bias of overreliance on clinical manifestations and endoscopic assessment .
collectively , our data showed similar and different expression patterns of traf4 and traf6 in patients with ibd . to date
 in recent years , interests combining the exploration of tumor necrosis factor receptor - associated factor 4 ( traf4 ) and traf6 in immune cells and transgenic mice are emerging . 
 although it has been found that traf4 and traf6 share the same traf binding sites , comprehensive study of traf4 and traf6 in inflammatory bowel disease ( ibd ) is still lacking . 
 this paper shows similar and different expression patterns of traf4 and traf6 in patients with ibd . 
 the results indicate that traf4 and traf6 are overexpressed in ibd . 
 traf4 and traf6 play different roles in the pathogenesis of ibd . 
 moreover , traf4 may be an indicator of endoscopic disease activity of uc and traf6 preactivation can be detected in noninflamed colonic segments . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion