NF-κB-dependent synergistic regulation of CXCL10 gene expression by IL-1β and IFN-γ in human intestinal epithelial cell lines Background and aims Little is known about the intestinal epithelial expression and secretion of CXCL10 -LRB- IP-10 -RRB- , a chemokine involved in recruiting T cells and monocytes . We aimed to study CXCL10 gene expression and regulation by the pro-inflammatory cytokines interleukin -LRB- IL -RRB- -1 β , interferon -LRB- IFN -RRB- - γ and tumour necrosis factor -LRB- TNF -RRB- - α in intestinal epithelial cell lines . Introduction Intestinal epithelial cells -LRB- IECs -RRB- form the first immunologic barrier of the organism while the villus surface is continuously in contact with various agents . IEC gene expression is tightly regulated to induce appropriate responses to pathogens and to avoid deleterious reactions to microbial flora . When stimulated with pro-inflammatory cytokines or infected with microbial pathogens , IECs may secrete chemokines , a set of low-molecular weight cytokines , directing migration and activation of leucocytes , which play a major role in the perpetuation of inflammatory processes -LSB- 1 -- 5 -RSB- . The final composition of leucocytes present in the inflamed intestine is most likely due to both secreted chemokines and the relative expression of chemokine cell surface receptors on different cell types . Chemokine production by colonic epithelial cells is thought to contribute to the characteristic increased infiltration of selected populations of leucocyte cells in inflammatory bowel disease -LRB- IBD -RRB- -LSB- 6 , 7 -RSB- . CXCL10 is a CXC chemokine , known to be secreted by endothelial cells -LSB- 8 -RSB- , keratinocytes -LSB- 9 -RSB- , eosinophils -LSB- 10 -RSB- and neutrophils -LSB- 11 -RSB- . It is also constitutively expressed by normal human colon epithelium -LSB- 12 -RSB- and chemoattracts activated T cells as well as monocytes by binding to the CXCR3 receptors present on the respective cells -LSB- 13 , 14 -RSB- . While CXCL10 up-regulation may occur in acute inflammation in response to pathogens , it is also known to be permanently over-expressed in IBD patients -LSB- 15 , 16 -RSB- . Blocking CXCL10 could prevent mice from acute colitis , suggesting this chemokine to play an important role in IBD pathogenesis -LSB- 17 -RSB- . Moreover , CXCL10 is believed to possess anti-tumoural properties by promoting anti-tumour T cell immunity -LSB- 18 , 19 -RSB- . Knowledge of its regulation in colonic cell lines may therefore give also insight into the pathobiology of colorectal cancer . Most previous studies on chemokine expression in IEC concentrated on CXCL8 -LRB- interleukin -LSB- IL -RSB- -8 -RRB- , a neutrophil chemoattractant -LSB- 20 , 21 -RSB- , but there were only few studies regarding the expression and secretion of CXCL10 in IECs -LSB- 12 -RSB- . Although it is well known that interferon -LRB- IFN -RRB- - γ is a major inducer of CXCL10 and that CXCL10 expression may be enhanced by pathogens and other inflammatory cytokines , the relative contribution of cytokines on CXCL10 induction , their synergistic action and the kinetics of cytokine-induced CXCL10 expression in intestinal epithelial cells has not been fully elucidated . Especially , the role of IL-1β in CXCL10 gene regulation in IECs and also in other cell types has not been fully evaluated so far . Intestinal mucosa is composed of cell populations in continuous change from a proliferative and undifferentiated stage in the basal parts of crypts to mature surface villus epithelial cells -LSB- 22 , 23 -RSB- . Little is known about the alteration of immunological functions as IECs mature . It has been shown that cellular differentiation affects IEC responsiveness to IL-1β -LSB- 24 -RSB- . In the present study , we aimed to elucidate the role of the pro-inflammatory cytokines IL-1β , tumour necrosis factor -LRB- TNF -RRB- - α and IFN-γ , which have established role in IBD pathogenesis , in inducing CXCL10 gene expression in IEC lines . Especially , the role of IL-1β , one of the most abundantly expressed cytokines , both in normal and inflamed mucosa -LSB- 25 -RSB- , in inducing CXCL10 expression by IECs and its possible signalling mechanisms was given importance . To consider possible influences of cellular differentiation on cytokine responsiveness , we performed studies on three different colonic epithelial cell lines . Our findings will get an insight into the role of intestinal epithelium in immune responses and in evaluating pathways , which might be targets of present and future pharmacotherapy of IBD and possibly colorectal cancer . Materials and methods Biological reagents The recombinant cytokines IL-1β , TNF-α and IFN-γ were purchased from Roche applied science -LRB- Mannheim , Germany -RRB- . An enzyme-linked immunosorbent assay -LRB- ELISA -RRB- kit for CXCL10 was purchased from RnD systems -LRB- Weisbaden , Germany -RRB- . The nuclear factor -LRB- NF -RRB- - κB inhibitor BAY11-7082 was purchased from Calbiochem -LRB- San Diego , CA -RRB- . Cell culture and stimulation protocols The human colon adenocarcinoma cell lines Caco-2 , HT-29 and DLD1 were obtained from DSMZ -LRB- Braunschweig , Germany -RRB- . Caco-2 cells were grown in Eagle 's minimal essential medium -LRB- BioWhittacker , Germany -RRB- containing 20 % foetal calf serum -LRB- FCS -RRB- supplemented with 100 U/ml each of penicillin and streptomycin and 1 % non-essential amino acids at 37 °C and 5 % CO2 . HT29 and DLD1 were grown in Roswell Park Memorial Institute medium containing 10 % FCS and 100 U/ml penicillin and streptomycin at 37 °C and 5 % CO2 . Caco-2 , HT29 and DLD-1 cells were plated into six-well plates at a density of 5 × 105 cells per well for real-time polymerase chain reactions -LRB- PCRs -RRB- , ELISA , Northern blotting and electrophoresis mobility shift assay -LRB- EMSA -RRB- experiments unless until stated and grown till they reached 70 -- 80 % confluence . These cells were then stimulated with IL-1β -LRB- 0.1 , 0.5 , 1 , 2 , 10 and 50 ng/ml -RRB- , TNF-α -LRB- 2 , 10 and 50 ng/ml -RRB- and IFN-γ -LRB- 2 , 10 and 50 ng/ml -RRB- based on the type of experiments . RNA isolation , real-time PCR Ribonucleic acid -LRB- RNA -RRB- was isolated by using Qiagen RNeasy mini kit according to the manufacturer 's protocol . The RNA concentrations were determined photometrically using a Gene Quant RNA/deoxyribonucleic acid -LRB- DNA -RRB- calculator -LRB- Pharmacia , Freiburg , Germany -RRB- . RNA was subsequently used for real-time PCR and Northern blot analysis . Reverse transcription of messenger RNA -LRB- mRNA -RRB- was performed using 1 μg of total cellular RNA . To determine the mRNA expression of CXCL10 , real-time PCR was carried out using gene-specific primers for human CXCL10 -LRB- Invitrogen GmbH , Karlsruhe -RRB- forward 5 ′ - CCA GAA TCG AAG GCC ATC AA-3 ′ , reverse 5 ′ - CAT TTC CTT GCT AAC TGC TTT CAG-3 ′ and β-actin -LRB- Invitrogen GmbH -RRB- forward 5 ′ - CTG GCA CCC AGC ACA ATG-3 ′ , reverse 5 ′ - CCG ATC CAC ACG GAG TAC TTG-3 ′ in an ABI Prism 7000 sequence detection system . PCR reaction was set up with Sybr ® Green PCR Master mix -LRB- Invitrogen -RRB- containing 0.3 μmol/l primers each and 1 μl of RT-product in 25 μl volume . A two-step amplification protocol was chosen consisting of initial denaturation at 95 °C for 10 min followed by 45 cycles with 15 s denaturation at 95 °C and 30 s annealing/extension at 60 °C . Finally , a dissociation protocol was performed to control specificity of amplification products . Relative expression of CXCL10 was then calculated using the comparative threshold-cycle -LRB- CT -RRB- method . The amount of target mRNA in each sample was normalized to the amount of β-actin mRNA designated as calibrator , to give ΔCT -LRB- CT β-actin − CT CXCL10 -RRB- . The relative expression of CXCL10 was calculated as the . MRNA expression is presented as fold increase calculated in relation to unstimulated cells after normalization to β-actin . Transient transfections Caco-2 , HT29 and DLD1 cells were plated in 24-well plates , 24 h before transfection -LRB- Costar , Corning , NY -RRB- , at a density of 50,000 / well in 1 ml medium . After 24 h , the cells were transfected with 250 ng of respective plasmids along with 20 ng of renilla plasmid using the non-liposomal formulation FuGENE -LRB- Roche Molecular Biochemicals , Mannheim , Germany -RRB- . On the day of transfection , fresh medium was added . FuGENE was added to the plasmid DNA at a ratio of 3 μl/μg DNA . FuGENE was prediluted in 100 μl serum-free medium and added drop-wise to the concentrated plasmid DNA . After 15 -- 25 min at room temperature , the mixture was added to the cells . The plasmids TGL-IP10 , TGL-IP10 κB2 Mut were kind gifts from Dr. Richard M. Ranshoff -LRB- Cleveland , OH -RRB- and were described elsewhere -LSB- 26 -RSB- . Luciferase assays Twenty-four hours after transfection , cells were stimulated with IL-1β -LRB- 0.1,1 , 2 , 10 and 50 ng/ml -RRB- , TNF-α -LRB- 2 , 10 and 50 ng/ml -RRB- , IFN-γ -LRB- 2 , 10 and 50 ng/ml -RRB- , IL-1β -LRB- 1 ng/ml -RRB- + TNFα -LRB- 50 ng/ml -RRB- , IL-1β -LRB- 1 ng/ml -RRB- + IFN-γ -LRB- 50 ng/ml -RRB- and TNFα -LRB- 50 ng/ml -RRB- + IFN-γ -LRB- 50 ng/ml -RRB- for 24 h , harvested and lysed , and luciferase was assayed by using a luciferase kit -LRB- Promega -RRB- . Values were normalised to renilla luciferase . Enzyme-linked immunosorbent assay To study the synergistic effects of cytokines on CXCL10 secretion , supernatants were collected at 0 , 3 , 6 , 12 and 24 h in Caco-2 and HT29 cells stimulated with IL-1β -LRB- 1 ng/ml -RRB- , TNF-α -LRB- 50 ng/ml -RRB- , IFN-γ -LRB- 50 ng/ml -RRB- , IL-1β -LRB- 0.5 ng/ml -RRB- + TNFα -LRB- 50 ng/ml -RRB- , IL-1β -LRB- 0.5 ng/ml -RRB- + IFN-γ -LRB- 50 ng/ml -RRB- and TNFα -LRB- 50 ng/ml -RRB- + IFN-γ -LRB- 50 ng/ml -RRB- and stored at − 70 °C until measured . CXCL10 protein secretion was measured by ELISA according to the manufacturer 's protocol . Preparation of nuclear extract Caco-2 cells were grown for 24 h , pre-treated for 1 h with BAY 11-7082 -LRB- 10 μM -RRB- and then stimulated with respective cytokines for 30 min . Cells were washed with 10 ml cold phosphate-buffered saline -LRB- PBS -RRB- twice , harvested , centrifuged at 1,000 × g for 5 min and resuspended in 1 ml of cold PBS . Centrifuged briefly , excess PBS was removed , and the pellet was resuspended in ice-cold extraction buffer -LRB- 20 mM hydroxyethyl piperazineethanesulfonic acid -LSB- HEPES -RSB- pH 7.8 , 10 mM KCL , 0.1 mM ethylenediamine tetraacetic acid -LSB- EDTA -RSB- , 1 mM dithiothreitol and 0.5 mM phenylmethanesulphonylfluoride -LSB- PMSF -RSB- -RRB- by gentle pippeting . Cells were then kept on ice for 15 min , and 30 μl of 10 % Nonidet NP-40 was added and vortexed briefly for 10 s . The lysed cell suspension was centrifuged for 30 s , and the supernatant was discarded . The pellet was resuspended in 50 μl extraction buffer containing 400 mM NaCl , agitated vigorously for 15 min at 4 °C . Then , the cell suspension was centrifuged for 5 min at 4 °C , and the supernatant containing the nuclear extract was collected and stored at − 80 °C until further use . Electrophoretic mobility shift assay and super-shift assay For binding reactions , 5 μg of nuclear extracts were incubated in 20 μl reaction mixture containing 40 mM HEPES pH 7.5 , 50 mM NaCl , 1 mM EDTA , 1mM DithitreitolHSCl , 1 mM PMSF , 1.28 μg/ml pBluescript vector DNA as half of the reaction and the rest being nuclear extract plus other components . Oligonucleotide duplex probes -LRB- Promega -RRB- were end-labelled with T4 polynucleotide kinase and -LSB- γ32P -RSB- adenosine triphosphate ; 1 × 104 cpm were then added to the reaction mixtures and incubated overnight at 4 °C . Reaction products were analysed by non-denaturing electrophoresis in a 5 % polycralamide gel with 0.5 × Tris -- borate -- EDTA buffer at room temperature . Gels were then exposed directly to X-ray film at − 70 °C for autoradiography . For competition , unlabelled oligonucleotides were added in molar excess at room temperature for 15 min before adding radiolabelled probe . In super shift experiments , nuclear extracts were incubated with anti-p65 anti-body -LRB- Santa Cruz Biotechnology , Santa Cruz , CA -RRB- overnight at 4 °C . Statistical analysis Statistical analyses were carried out using the Prism software packet -LRB- Version 3.0 , Graphpad Software , San Diego , CA -RRB- . Comparisons between two or more different treatment groups were made with the non-paired t test or analysis of variance -LRB- ANOVA -RRB- where appropriate . For time kinetics experiments , a two-way ANOVA test followed by the Bonferroni post-test was performed . In case of RNA expression , a log transformation was performed before . Statistical differences were regarded significant at a p level below 0.05 . Data were expressed as means ± standard error of the mean -LRB- SEM -RRB- . Results Differential expression of CXCL10 mRNA in intestinal epithelial cell lines under basal conditions In the present study , the basal mRNA expression of CXCL10 in three different IECs , Caco-2 , HT29 and DLD1 cells , were studied first . Real-time PCR experiments using gene-specific primers for CXCL10 revealed that the basal mRNA expression of CXCL10 was highest in Caco-2 followed by HT29 and DLD1 -LRB- Fig. 1 -RRB- . Fig. 1Basal level expression of CXCL10 in IECs . Caco-2 , HT29 and DLD1 cells were plated at a density of 5 × 105 cells in six well plates , grown for 24 h and total RNA from the cells was isolated . First-strand complementary DNA was prepared from 1 μg of total RNA . Real-time PCR was performed for CXCL10 and β-actin was used as an internal control . The bars indicate the mean of six individual experiments . Asterisk corresponds to p < 0.01 IL-1β , TNF-α and IFN-γ dose-dependent influence on CXCL10 gene expression in IECs Caco-2 , HT29 and DLD1 cells were tested for their ability to respond to pro-inflammatory cytokines by the induction of CXCL10 mRNA expression . Cells were stimulated for 4 h with various concentrations of cytokines , and CXCL10 expression was analysed by real-time PCR . In response to IFN-γ and TNF-α , Caco-2 , HT29 and DLD1 cells showed a dose-dependent induction of CXCL10 mRNA -LRB- Fig. 2 -RRB- . Increased expression of CXCL10 mRNA was observed when 2 ng/ml IL-1β was used , while a higher concentration did not further enhance expression . Further , we checked the response of IECs to lower doses of IL-1β showing a dose response in the range of 0.1 to 1 ng/ml in the induction of CXCL10 mRNA . IL-1β at a concentration of 0.5 ng/ml was able to induce maximum expression of CXCL10 mRNA at 4 h -LRB- data not shown -RRB- . Fig. 2Dose-dependent expression of CXCL10 mRNA in IECs stimulated with cytokines . 5 × 105 cells were plated into six-well plates and grown for 24 h and then stimulated with IL-1β , TNF-α and IFN-γ at an increasing doses of 2 , 10 and 50 ng/ml . The cells were harvested after 4 h , total RNA from the cells was isolated , and first-strand cDNA was prepared from 1 μg of total RNA . Real-time PCR was performed for CXCL10 , and β-actin was used as an internal control . Graph represents the mean ± SEM of four individual experiments . All cytokine-stimulated expressions were significantly different from unstimulated controls -LRB- p < 0.05 -RRB- . Asterisk , no significant difference between different IL-1β concentrations -LRB- p < 0.05 -RRB- IL-1β , TNF-α and IFN-γ time-dependent influence on CXCL10 gene expression in IECs Once we confirmed the responsiveness of IECs to pro-inflammatory cytokines , we next examined the mRNA kinetics of CXCL10 induced by the cytokines alone or in combination with each other . Each cell line had a different time course of response when stimulated with respective cytokines and their combinations -LRB- Fig. 3a -- c -RRB- . In Caco-2 , stimulated with IL-1β or TNF-α , CXCL10 mRNA was induced within the first 40 min , and the induction reached to a peak after 4 h , while IFN-γ had a later initial effect inducing CXCL10 mRNA after 2 h and reached to a maximum at 8 h. Combination of IL-1β + IFN-γ had a stronger synergistic effect in the induction of CXCL10 mRNA within 20 min and reaching to maximum after 4 h , whereas TNF-α + IFN-γ synergistically led to a later induction of CXCL10 mRNA starting at 40 min but had a strong synergistic effect after 8 h when compared to IL-1β + IFN-γ -LRB- Fig. 3a -RRB- . In HT29 -LRB- Fig. 3b -RRB- and DLD1 -LRB- Fig. 3c -RRB- , CXCL10 mRNA time kinetics deviated from that in Caco-2 -LRB- Fig. 3a -RRB- . CXCL10 mRNA was induced after 1 h of stimulation with the respective single cytokines in DLD1 . In case of HT29 , either TNF-α or IFN-γ alone led to delayed induction of CXCL10 mRNA after 1 h , whereas IL-1β alone led to an induction starting at 2 h. HT29 and DLD1 cells treated with combinations of IL-1β + IFN-γ showed a strong synergistic induction of CXCL10 mRNA within 40 min , whereas the TNF-α + IFN-γ combination led to an even stronger induction of CXCL10 mRNA within 20 min in HT29 and within 40 min in DLD1 . IL-1β + TNFα in combination had no synergistic effect on CXCL10 mRNA induction in all the cell lines used for this study . The real-time PCR results could be confirmed by Northern blot analysis in Caco-2 and HT29 cells . However , the detection threshold was not low enough to display basal and single cytokine-induced CXCL10 mRNA -LRB- data not shown -RRB- . Fig. 3Time kinetics of CXCL10 mRNA expression in IECs stimulated with cytokines . a Caco-2 , b HT29 and c DLD1 cells were plated at a density of 5 × 105 cells in six-well plates and grown for 24 h and then stimulated with respective cytokines and their combinations . Cells were harvested at different time points as indicated , and total RNA from the cells was isolated and first-strand cDNA was prepared from 1 μg of total RNA . Real-time PCR was performed for CXCL10 , and β-actin was used as an internal control . Graphs represent the mean ± SEM of three to five experiments . IFN-γ led to a significant increase in CXCL10 expression from 2 h onwards . A significant increase -LRB- p < 0.05 -RRB- from control was observed in all expect in those marked as N.S. -LRB- non-significant -RRB- . Number sign indicates time points with significant differences -LRB- p < 0.05 -RRB- of cytokine combinations or IL-1β compared to IFN-γ alone IL-1β , TNF-α and IFN-γ time-dependent influence on CXCL10 protein secretion in IECs To test whether cytokine stimulation of IECs would lead not only to the induction of CXCL10 mRNA but also to the synthesis and secretion of the protein , Caco-2 , HT29 and DLD1 cells were incubated with respective cytokines -LRB- from 3 to 24 h -RRB- , and secretion of CXCL10 was detected by ELISA . Under basal conditions , Caco-2 cells secreted 0.012 ± 0.003 ng/ml CXCL10 . Treatment with IL-1β , TNF-α and IFN-γ led to a time-dependent increase in CXCL10 protein , with IL-1β being the most potent stimulus during the first 6 h -LRB- Fig. 4a -RRB- . Of importance , after 12 h , CXCL10 secretion was stimulated strongly by IFN-γ . Co-incubation of Caco-2 with respective cytokines revealed a synergistic effect of IL-1β + IFN-γ and TNF-α + IFN-γ on CXCL10 secretion , reaching levels of 3.354 ± 0.690 and 7.808.1 ± 0.173 ng/ml after 24 h . The combination of IL-1β + TNF-α had no synergistic effect on CXCL10 secretion when compared to the respective cytokines alone . These time kinetic studies revealed that in Caco-2 , IL-1β alone or in combination with IFN-γ induced the secretion of CXCL10 very early compared to the combination of TNF-α + IFN-γ , which induced a delayed but stronger synergistic secretion of CXCL10 protein , suggesting that in case of IL-1β + IFN-γ , a synergism could take place very early , e.g. , at the level of intracellular signal cascades . In HT29 cultures , CXCL10 secretion was not detectable under basal condition . TNF-α or IFN-γ alone led to an induction of CXCL10 protein secretion within 3 h -LRB- 0.0172 ± 0.005 and 0.011 ± 0.001 ng/ml -RRB- , whereas IL-1β could not induce the secretion of CXCL10 protein . IL-1β , TNF-α and IFN-γ led to a time-dependent increase in CXCL10 concentrations in HT29 cell supernatants reaching maximum levels of 0.039 ± 0.0057 , 0.632 ± 0.0222 and 19.929 ± 0.838 ng/ml showing that IFN-γ is the strongest inducer of CXCL10 protein secretion when compared to other two cytokines in this cell line -LRB- Fig. 4b -RRB- . The combinations of TNFα + IFN-γ and IL-1β + IFN-γ induced CXCL10 protein secretion to 1.822 ± 0.109 and 0.410 ± 0.027 ng/ml within 3 h and led to a maximum of 492.728 ± 83.776 and 87.158 ± 7.913 ng/ml after 24 h -LRB- Fig. 4b -RRB- . Fig. 4Time kinetics of CXCL10 protein secretion in Caco-2 and HT29 cells stimulated with cytokines . 5 × 105 cells were plated into six-well plates and grown for 24 h and then stimulated with IL-1β , TNF-α and IFN-γ as indicated . Supernatants were collected at respective time points from a Caco-2 and b HT29 cells and ELISA for CXCL10 protein was performed . Data represent means of three to five experiments ± SEM done in duplicates for each sample . Asterisk indicates time points with significant differences -LRB- p < 0.05 -RRB- of cytokine combinations compared to IFN-γ alone CXCL10 promoter activation by cytokines TGL-IP10 , a promoter-reporter containing 435 bp of human CXCL10 sequence upstream of the transcriptional start site , was used to evaluate the role of IL-1β , TNF-α and IFN-γ alone or in combination with each other in the transcription of CXCL10 . When TGL-IP10-transfected Caco-2 cells were stimulated with single cytokines , IL-1β -LRB- 1 ng/ml -RRB- led to a stronger induction -LRB- 6.53 ± 1.65-fold increase -RRB- than TNF-α -LRB- 50 ng/ml -RRB- and IFN-γ -LRB- 50 ng/ml -RRB- alone -LRB- 2.76 ± 0.53 and 3.14 ± 1.2-fold increase -RRB- . The combination of IL-1β + IFN-γ showed a stronger synergistic induction -LRB- 26.18 ± 11.68 -RRB- than the combination of TNF-α + IFN-γ -LRB- 13.97 ± 3.25-fold increase -RRB- -LRB- Fig. 5a -RRB- . In HT29 cells , when stimulated with single cytokines , IFN-γ -LRB- 50 ng/ml -RRB- led to a maximum induction -LRB- 6.52 ± 0.58-fold increase -RRB- , followed by TNF-α -LRB- 4.44 ± 0.16-fold increase -RRB- and IL-1β -LRB- 1.43 ± 0.05-fold increase -RRB- . The combination of IL-1β + IFN-γ synergistically led to 12.76 ± 3.21-fold induction , and TNF-α + IFN-γ led to 26.70 ± 7.5-fold induction of promoter activity -LRB- Fig. 5b -RRB- . In DLD1 cells -LRB- Fig. 5c -RRB- , IFN-γ -LRB- 50 ng/ml -RRB- led to a maximum induction -LRB- 8.06 ± 0.51-fold increase -RRB- of CXCL10 promoter activity followed by TNF-α -LRB- 50 ng/ml -RRB- induction -LRB- 3.59 ± 0.08-fold increase -RRB- and IL-1β -LRB- 1 ng/ml -RRB- induction -LRB- 2.32 ± 0.12-fold increase -RRB- . The combination of IL-1β + IFN-γ led to a 40.72 ± 12.19-fold induction , and TNF-α + IFN-γ led to a 20.56 ± 6.27 fold induction . In all three cell lines , the combination of IL-1β + TNF-α had no synergistic effect on CXCL10 promoter activity . Fig. 5Transient transfection analysis of TGL-IP10 in IECs . The CXCL10 promoter containing reporter construct , pTGL-IP10 and pRL-TK -LRB- renilla -RRB- plasmids , was transiently transfected into a Caco-2 , b HT29 and c DLD1 cells . 24 h after transfection , cells were stimulated with cytokines as indicated for 24 h , harvested , and promoter activity was monitored by luciferase assay . Data were normalised with renilla luciferase values and included as fold increase vs control . Data represent the means ± SEM of three to five independent experiments done in duplicates for each sample . Asterisk indicates an induction significantly different -LRB- p < 0.05 -RRB- from control -LRB- C -RRB- . Significant differences between combinations of cytokines and the respective single cytokines are indicated with bars with a number sign -LRB- p < 0.05 -RRB- . N.S. is not significant Evaluating the role of NF-κB in CXCL10 gene expression To evaluate the possible role of NF-κB in cytokine-induced CXCL10 gene expression , experiments with a specific NF-κB inhibitor were performed . The effect of BAY 11-7082 on NF-κB inhibition was evaluated by reporter assays . BAY11-7082 was able to dose-dependently inhibit IL-1β-induced pNF-κB-SEAP gene reporter activity in Caco-2 cells -LRB- data not shown -RRB- . Based on this experiment , we could determine that 10 μM of BAY11-7082 was sufficient to inhibit the NF-κB activation . The induction of CXCL10 gene expression by cytokines was inhibited by addition of the NF-κB-specific inhibitor BAY11-7082 in Caco-2 , HT29 and DLD1 cells , with a varying degree of inhibition in the individual cell lines . Inhibition of NF-κB led to almost complete inhibition -LRB- approximately 90 -- 95 % inhibition -RRB- of CXCL10 mRNA and protein secretion induced by cytokines in Caco-2 and DLD1 cells -LRB- Fig. 6a and c -RRB- . In DLD1 cells , IL-1β -LRB- 1 ng/ml -RRB- did not have any inducing effect on CXCL10 secretion . In HT29 , inhibition of NF-κB had approximately 60 -- 75 % inhibitory effect on cytokine-induced CXCL10 mRNA expression and secretion -LRB- Fig. 6b -RRB- . Fig. 6Role of NF-κB in cytokine-induced CXCL10 mRNA expression by IECs . 5 × 105 cells were plated and grown for 24 h. Then , the cells were pre-treated with 10 μM of BAY 11-7082 for 1 h and then stimulated with respective cytokines and their combinations as stated in the figure for 2 and 24 h . After 2 h , cells were harvested for RNA isolation to perform real-time PCRs , and at 24 h , cell supernatants were collected and stored at − 70 °C until ELISA was performed for CXCL10 . Data presented are the means ± SEM of three independent experiments done in duplicates for each sample in a Caco-2 , b HT29 and c DLD1 cells . Significant CXCL10 inhibition by BAY 11-7082 is indicated by an asterisk -LRB- p < 0.05 -RRB- . N.S. is not significant To further evaluate the role of NF-κB , transient transfections were done with a CXCL10 promoter construct bearing a mutated NFkB-binding site -LRB- TGL-IP10 κB2 Mut -RRB- . In Caco-2 cells , TGL-IP10 κB2 Mut was not responsive to any of the cytokines and their combinations , suggesting that CXCL10 gene induction is strongly dependent on NF-κB in this cell line -LRB- Fig. 7a -RRB- . In HT29 and DLD1 cells , the combination of TNF-α + IFN-γ led to 6.49 ± 2.13 and 4.87 ± 1.43-fold increase , and the combination of IL-1β + IFN-γ led to 2.26 ± 0.63 and 4.75 ± 0.53-fold increase of the TGL-IP10 κB2 Mut promoter activity -LRB- Fig. 7b and c -RRB- , revealing that a smaller portion of CXCL10 gene expression might be regulated independently from NF-κB . Fig. 7Transient transfection analysis of TGL-IP10 and TGL-IP10 κB2 mutant in IECs . TGL-IP-10 and TGL-IP10-κB2 Mut plasmid was transiently transfected into a Caco-2 , b HT29 and c DLD1 . 24 h after transfection , cells were stimulated with cytokines for 24 h , and promoter activity was monitored by luciferase assay . Data were normalised with renilla luciferase values . Data presented are the means ± SEM of three to five independent experiments done in duplicates for each sample . Significant differences vs controls -LRB- C -RRB- are indicated by an asterisk -LRB- p < 0.05 -RRB- . Significant differences of wild type vs mutant are indicated by a number sign -LRB- p < 0.05 -RRB- . N.S. is not significant To confirm further our results from promoter and inhibitor studies , we did gel-shift and super shift assays to show that NF-κB is activated by cytokines and that the activated NF-κB complex contains the NF-κB functional sub-unit p65 . In the nuclear extracts of Caco-2 cells , we could clearly see a shift in the samples treated with IL-1β , TNF-α alone or the combinations of IL-1β + IFN-γ and TNF-α + IFN-γ . The protein-DNA complex was efficiently competed by unlabelled κB oligonucleotides . There was no shift in the samples treated with the NF-κB inhibitor BAY11-7082 , stating that the shift was indeed because of NF-κB binding . Furthermore , antibody super shift assay using anti-p65 antibody had clearly shown that the complex contains NF-κB functional sub-unit , p65 -LRB- Fig. 8 -RRB- . Fig. 8Gel shift and super shift analysis for NF-κB . Caco-2 cells were seeded and grown for 24 h before pre-treating with BAY 11-7082 -LRB- 10 μM -RRB- for 1 h and then stimulated with respective cytokines for 30 min ; cells were then harvested , and nuclear extracts were prepared . 5 μg of nuclear extracts were used for gel shifts with a wild-type κB oligonucleotide . Super shifts were performed with anti-p65 antibody . Similar results were obtained in two separate experiments Discussion In addition to its many homeostatic functions such as barrier protection , mucus production and resorption , the intestinal epithelium plays an important role in regulating local inflammation and immune responses . The intestinal epithelium participates in inflammatory responses in part through the generation of numerous cytokines and chemokines that mediate recruitment and activation of inflammatory cells . The epithelium is involved in local cytokine networks allowing first response to noxious agents as well as cross-talk with immune cells to help provide an effective inflammatory response . These bi-directional inflammatory communications are necessary for normal host defence , but they also play an important role in the pathogenesis of IBD and might be also of importance in modulating anti-tumoural immunity in colonic malignancies . In this report , we demonstrate that pro-inflammatory cytokines , which may be produced by sub-mucosal immune cells , can differentially and co-ordinately regulate CXCL10 gene expression in IECs . CXCL10 is one of several chemokines produced by IEC . Previous studies could show that IFN-γ in combination with TNF-α caused induction of RANTES -LRB- regulated upon activation , normal t-cell expressed , and secreted protein -RRB- and monocyte chemotactic protein-1 in colorectal tumour cell lines , while IFN-γ had no effect on CXCL8 -LRB- IL-8 -RRB- secretion -LSB- 27 -RSB- , the latter observation being confirmed in all three cell lines in the present study -LRB- data not shown -RRB- . Dwinell et al. -LSB- 12 -RSB- had previously shown that stimulation of IECs with TNF-α or IL-1α strongly potentiated IFN-γ-induced CXCL10 . It is well known that among IL-1α and IL-1β , the latter is secreted and has an active role in inflammation . Importantly , IL-1β was shown to be over-expressed in IBD patients -LSB- 25 , 28 -RSB- . This makes IL-1β an interesting cytokine to study its effect on CXCL10 gene regulation . Although the synergistic effects of TNF-α + IFN-γ on CXCL10 gene expression and the signalling mechanisms involved were studied in astrocytes -LSB- 26 -RSB- and their synergistic effect on inducible nitric oxide synthase expression in IECs and macrophages -LSB- 29 -RSB- had been studied in detail , the synergistic action of these cytokines on CXCL10 gene expression and the signalling mechanisms involved have not been evaluated in IECs . Especially , the role of IL-1β alone and its synergism with both TNF-α and IFN-γ on CXCL10 gene expression in IECs has been given no attention . Previously , it has been shown that IECs may express elevated levels of CXCL10 -LSB- 12 -RSB- , and its expression is increased in the mucosa of patients with IBD -LSB- 16 -RSB- . A critical role for CXCL10 in IBD has also been shown in animal models -LSB- 30 , 31 -RSB- . Apart from this , CXCL10 has been shown to be expressed constitutively in colorectal carcinomas and primary metastatic sites exerting anti-tumour activity by recruiting mononuclear cells via the CXCR3 receptor . However , at the same time , it has also been shown that CXCL10 promoted metastatic adhesion and tumour cell migration -LSB- 32 -RSB- so that care should be taken considering CXCL10 as an anti-tumoural therapy option . In light of these findings , we assessed whether stimulation of IECs in culture by inflammatory mediators could induce CXCL10 gene expression . All three IEC lines we used expressed CXCL10 mRNA in a concentration-dependent manner when stimulated with IFN-γ and TNF-α alone . These data support a vast body of evidence that demonstrates that IFN-γ can induce CXCL10 expression in a variety of cells types , including granulocytes , T lymphocytes , bronchial epithelial cells and keratinocytes -LSB- 33 , 34 -RSB- , while TNF-α is a potent inducer for CXCL10 in hepatocytes -LSB- 17 -RSB- . Importantly , in addition to these data , we could also show that Caco-2 cells stimulated with IL-1β up-regulate CXCL10 mRNA to a significant level , whereas in HT29 and DLD1 cells , induction of CXCL10 mRNA was less pronounced . A number of authors have reported functional synergy between TNF-α and IFN-γ in promoting gene expression for proteins such as RANTES , IFN regulatory factor 1 -LRB- IRF-1 -RRB- , MIG and intercellular adhesion molecule-1 -LSB- 35 -- 38 -RSB- in a variety of cell types and have demonstrated that this synergy depends on the co-existence of TNF-α-responsive NF-κB-binding sites and IFN-γ-responsive signal transducers and activators of transcription -LRB- STAT -RRB- protein-binding elements within the promoters of the genes of interest -LSB- 35 , 36 -RSB- . In our study , when IFN-γ and TNF-α were combined , the accumulation of CXCL10 mRNA and protein was elevated compared to unstimulated cells or cells stimulated with either cytokine alone , implying a synergistic effect on transcription of the CXCL10 gene . A synergistic effect by these two cytokines on CXCL10 expression was also demonstrated in astrocytes , and this synergy has been shown to be at least in part dependent on NF-κB activation and binding to a NF-κB-binding site within the CXCL10 promoter -LSB- 26 -RSB- . The synergistic action of IL-1β with IFN-γ in IECs has not been characterised so far . Recently , Takami et al. -LSB- 39 -RSB- showed the synergistic induction of hepatocyte growth factor in human skin fibroblasts by IL-1β + IFN-γ + IFN-γ -LSB- 39 -RSB- . We here report for the first time that in intestinal epithelial cell lines , IL-1β alone or in combination with IFN-γ induced CXCL10 gene expression . In Caco-2 cells , the combination led to an early synergistic induction of both CXCL10 mRNA and protein compared to the combination of IFN-γ + TNF-α . This early induction of CXCL10 mRNA and protein by IL-1β + IFN-γ in Caco-2 might be due to the early convergence of signalling pathways that activates transcription factors like NF-κB , IRF-1 , STAT1 or other factors that mediate transcription factor interactions . Transient transfections with human CXCL10 promoter -LSB- 26 -RSB- , in Caco-2 , HT29 and DLD1 cells , showed that the synergistic effect of IL-1β + IFN-γ and IFN-γ + TNF-α on CXCL10 gene expression in these cell lines was indeed because of the transcriptional synergy employed at the level of the CXCL10 promoter . A number of studies had revealed that cytokines like IL-1β , TNF-α or enteroinvasive bacteria and the bacterial cell wall product lipopolysaccharide induce the expression of several inflammatory genes through the transcription factor NF-κB in IECs -LSB- 40 -- 43 -RSB- . To find out the signalling mechanism involved in CXCL10 gene expression in IECs , we explored the role of transcription factor NF-κB . Inhibition of NF-κB with BAY11-7082 clearly inhibited both CXCL10 mRNA and protein induced by single cytokine alone or in combinations in Caco-2 , HT29 and DLD1 cells . Gel shift in Caco-2 could confirm that NF-κB is activated and binds to consensus κB oligonucleotides in cells treated with either IL-1β or TNF-α alone and in combination with IFN-γ . It was shown previously that the binding complex of NF-κB to the κB2-binding site of the CXCL10 promoter contains the homodimer of p65 -LSB- 26 -RSB- . In our study , super shift assays in Caco-2 cells treated with IL-1β alone or in combination with IFN-γ confirmed the presence of p65 in the complexes bound to the consensus κB oligo nucleotides . In addition to these experiments , transient transfection experiments with a κB2 mutant of the CXCL10 promoter -LSB- 26 -RSB- clearly showed that in Caco-2 cells , none of the cytokines and their combinations were able to induce the CXCL10 promoter activity . In HT29 cells , the combination of IFN-γ + TNF-α led to a weaker induction of TGL-IP10κB-2 mutant activity , whereas in DLD1cells , the combination of IL-1β + IFN-γ and IFN-γ + TNF-α led to a weak induction of TGL-IP10 κB-2 mutant activity . Based on these data , we can conclude that NF-κB plays a critical role in CXCL10 gene expression induced by cytokines in IECs , and this is mainly dependent on the κB-2 site present in its promoter . Moreover , IFN-γ-induced CXCL10 mRNA and protein secretion was inhibited upon NF-kB inhibition . Although the present results may reflect only a small aspect in the overall concert of chemokines , it seems apparent that the basic regulatory principles elucidated here might be shared also by a number of other chemokine genes like that of CCL19 , which has recently been shown to be regulated by multiple NF-κB and IRF family transcription factors in human monocyte-derived dendritic cells -LSB- 44 -RSB- . Recently , Hiroi M et.al . -LSB- 45 -RSB- showed that co-operation between IFN-γ induced STAT1 , and the constitutively active or inducible NF-κB is necessary for the transcriptional activity of IFN-γ-inducible genes like MIG and CXCL10 . Therefore , we suggest a similar mechanism for IFN-γ-induced CXCL10 in IECs . The responsiveness to cytokines might depend on the state of differentiation . Schlottmann et . al. -LSB- 46 -RSB- recently showed that in contrast to HT29 where IFNγ up-regulated CXCL8 secretion , the same was unaffected in Caco-2 cells or even down-regulated in primary colonic epithelial cells . Future studies on chemokine regulation should therefore include primary cells , keeping well in mind that the presence of contaminating immune cells , culture conditions and cell stress during isolation may influence results . Taken together , our data suggest that while IFN-γ , TNF-α and IL-1β may work individually through disparate signalling pathways in CXCL10 gene regulation , a combination of these cytokines enhances transcription of the CXCL10 gene in an NF-κB-dependent manner . The fact that IL-1β via activation of NF-κB is able to mimic the effect of TNF-α in augmenting CXCL10 expression might be of importance in patients with IBD , who do not respond to anti TNF-α antibody therapy . In those cases , an additional inhibition of IL-1β might be desirable as a future therapy option in treating IBD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

intestinal epithelial cells
inflammatory bowel disease
cytokines and chemokines


Physiological importance of RNA and protein mobility in the cell nucleus Trafficking of proteins and RNAs is essential for cellular function and homeostasis . While it has long been appreciated that proteins and RNAs move within cells , only recently has it become possible to visualize trafficking events in vivo . Analysis of protein and RNA motion within the cell nucleus have been particularly intriguing as they have revealed an unanticipated degree of dynamics within the organelle . These methods have revealed that the intranuclear trafficking occurs largely by energy-independent mechanisms and is driven by diffusion . RNA molecules and non-DNA binding proteins undergo constrained diffusion , largely limited by the spatial constraint imposed by chromatin , and chromatin binding proteins move by a stop-and-go mechanism where their free diffusion is interrupted by random association with the chromatin fiber . The ability and mode of motion of proteins and RNAs has implications for how they find nuclear targets on chromatin and in nuclear subcompartments and how macromolecular complexes are assembled in vivo . Most importantly , the dynamic nature of proteins and RNAs is emerging as a means to control physiological cellular responses and pathways . Introduction It is an irony of science that the discovery of fundamental principles is often delayed . A case in point is the cell biological investigation of the cell nucleus . The nucleus is arguably the functionally most prominent cellular organelle as it houses the vast majority of the genetic information and is the exclusive site of essential processes including transcription , RNA processing , replication and DNA repair . Yet , our understanding of how the nucleus is organized and how nuclear architecture contributes to cellular function is only now emerging and still lags behind that of most other cellular compartments . Fortunately , pioneering studies over the past decade have uncovered several of the fundamental principles which determine nuclear architecture and function -LRB- Lanctot et al. 2007 ; Misteli 2001 ; Spector 2003 -RRB- . These studies have revealed the presence of distinct structural elements , particularly lamin proteins and possibly short actin filaments , within the nucleus and they have led to the identification of a large number of distinct nuclear subcompartments in which specific nuclear functions , such as the synthesis and processing of ribosomal RNA in the nucleolus , occur -LRB- Burke and Stewart 2006 ; Gruenbaum et al. 2005 ; Handwerger and Gall 2006 ; Hernandez-Verdun 2006 ; Lamond and Sleeman 2003 -RRB- -LRB- Fig. 1a -RRB- . In addition , it has become clear that the genome is non-randomly organized within the spatial confines of the nucleus with specific chromosomes and genes preferentially localizing to specific sites -LRB- Lanctot et al. 2007 ; Meaburn and Misteli 2007 -RRB- -LRB- Fig. 1b , c -RRB- . These observations paint a picture of a highly complex and heterogeneous cell nucleus . They also indicate that some properties of the nucleus are counterintuitive . For example , despite the requirement to accommodate ∼ 2 m of DNA within the cell nucleus of a diameter of typically ∼ 10 μm , chromatin is estimated to only occupy about 15 % of the nuclear volume . Fig. 1Nuclear architecture and function . The mammalian cell nucleus contains a large number of non-random and structural features . a Nuclear lamins -LRB- green -RRB- form a network of architectural elements at the periphery . This nuclear lamina is implicated in mechanical support of the nucleus , organization of chromatin -LRB- red/blue -RRB- in the nuclear interior , as a platform for signaling events and in the sequestration of nuclear proteins . b , c Genomes are non-randomly organized within the nucleus . b Each chromosome occupies a distinct chromosome territory -LRB- red chromosome 14 ; green chromosome 17 in human breast epithelial cells -RRB- . c Pericentromeric heterochromatin regions -LRB- white -RRB- from multiple chromosomes often cluster into chromocenters . Bar 6.3 μm . Image courtesy : a Gianluca Pegoraro , b Karen Meaburn , c Manjari Mazumdar , all National Cancer Institute , NIH A key question in understanding nuclear architecture is how protein and RNAs move within the nucleus . This is a key issue if we are to gain a full appreciation of what the nuclear environment is like . More importantly , the mode of nuclear motion of molecules has physiological implications as it affects how proteins find their target sequences in the genome , how RNAs are exported from the nucleus upon transcription and how proteins are sequestered within the nucleus for regulatory purposes . Some initial insights into these issues are summarized in broad terms here . The motion of RNA and proteins in the cell nucleus We have long known that proteins and RNAs must be able to move within the cell nucleus . Messenger RNAs traverse the nucleus and are promptly exported from the nucleus after their synthesis and complete processing . Newly synthesized proteins on the other hand are imported into the nucleus and rapidly disperse throughout its volume , implying effective intranuclear trafficking . Furthermore , proteins rapidly relocalize within the nucleus upon experimental or physiological changes in conditions . For example , inhibition of protein synthesis leads to rapid and dramatic relocalization of a large number of nucleolar proteins -LRB- Andersen et al. 2005 -RRB- , and pre-mRNA splicing factors swiftly respond to transcriptional activation and inhibition -LRB- Jiménez-García and Spector 1993 ; Misteli et al. 1997 -RRB- . How proteins and RNAs move within the nucleus and how they find their targets , however , was unknown for long . It was not clear whether movements are energy dependent , whether they occur by directed transport or what the speed of intranuclear trafficking is . These fundamental questions have recently been answered -LRB- Misteli 2001 -RRB- . The investigation of intranuclear protein and RNA dynamics was made possible by the development of techniques to visualize and measure the motion of these molecules by time-lapse microscopy . Tracking of proteins by photobleaching methods revealed a surprising degree and speed of intranuclear trafficking . The analysis of tracers such as GFP or dextran which do not undergo specific interactions with DNA or other proteins indicated that the nuclear environment is permissive for rapid diffusional motion -LRB- Görisch et al. 2005 ; Misteli 2001 ; Seksek et al. 1997 -RRB- . The diffusion of molecules within the nucleus is only limited by the steric constraints imposed by chromatin and nuclear bodies -LRB- Görisch et al. 2005 ; Misteli 2001 ; Seksek et al. 1997 -RRB- . The typically measured diffusion coefficients of 10 -- 100 μm2 s − 1 are similar to those observed in the cytoplasm and are only ∼ 5 times less than that in solution . As expected for diffusion , this rapid motion is energy-independent and non-directional . RNA motion within the nucleus is similarly rapid and non-directional . Several methodological approaches involving fluorescently labeled , microinjected , engineered or endogenous RNAs demonstrate that ribosomal RNAs as well as polyA-RNAs move freely in a non-directional manner with a diffusion coefficient of 0.03 -- 0.1 μm2 s − 1 within the nucleus -LRB- Ritland-Politz et al. 2006 ; Shav-Tal et al. 2004 -RRB- . A diffusion coefficient of this magnitude is sufficient to ensure transport of an RNA particle from deep within the nucleus to the cytoplasm within a few minutes , consistent with biochemical observations on kinetics of RNA maturation and transport . Thus energy-independent , diffusion-based movement of RNA particles alone can account for the observed kinetics of RNA export , and no active mechanisms are required to ensure rapid export . An impressive demonstration of the non-directional motion of mRNA comes from studies in which a nascent RNA is visualized at its site of transcription and its export to the cytoplasm measured in living cells -LRB- Ritland-Politz et al. 2006 ; Shav-Tal et al. 2004 -RRB- . These studies show that RNAs synthesized from genes positioned in proximity to the nuclear envelope diffuse away from their site of synthesis in all directions rather than follow a direct path to the nearest nuclear pore . This observation powerfully demonstrates the non-directed , diffusion-based motion of RNAs in the nucleus . Trafficking as a means of targeting One of the conceptually most challenging problems in cell biology is the question of how molecules find their specific targets within a cell or within an organelle . This problem is particularly complex in the cell nucleus where transcriptional regulators need to find their specific target genes amongst the myriad of potential binding sites within the genome . Somewhat counter-intuitively , the non-directional , but rapid , motion of proteins within the cell nucleus provides a means to ensure targeting of proteins to specific genome locations -LRB- Misteli 2001 -RRB- . The power of targeting by random diffusion within the nucleus is best illustrated when considering how a transcription factor finds its target genes but it also applies to targeting of proteins to nuclear compartments or any other nuclear site . Proteins a priori do not know where their targets are and we do know of any directed transport systems , such as a molecular motor-based mechanism , that would bring a factor to a specific gene or a specific location within the nucleus . Thus , the only way for a transcription factor to find its target is to scan the genome . The intrinsic ability of proteins to rapidly move within the nucleus by diffusion-based mechanisms permits such genome scanning -LRB- Misteli 2001 -RRB- . This occurs by a transcription factor freely diffusing within the nucleoplasm until it interacts by chance with chromatin . The molecule will now probe whether the sequence it has encountered is a specific binding site such as in the promoter of one of its target genes . If it is , the transcription factor will be captured and stably associated with its specific target site . If the sequence is not a binding site , the molecule , after a short interaction , will dissociate from chromatin and continue its diffusional journey through the nucleus . It might appear at first glance that such a stop-and-go model for genome scanning would be insufficiently effective . However , the observed dynamic properties of transcription factors are entirely consistent with this model . We know that the residence time of most transcription factors even on specific DNA binding sites is in the order of a few seconds and that their interaction with non-specific sites is even faster , most likely in the order of tens of milliseconds -LRB- Gorski et al. 2006 ; Houtsmuller et al. 1999 ; McNally et al. 2000 ; Phair et al. 2004 ; Sprague et al. 2006 -RRB- . Assuming these time scales for binding , one can calculate that it takes a single transcription factor molecule only a few minutes to search the entire genome space . Considering that most transcription factors exist in several thousand copies and have multiple target genes , their random diffusional motion is entirely sufficient to ensure a steady supply at their target genes . This assumption is further supported by direct measurement of transcription factor flux on an endogenous rRNA promoter demonstrating the collision of several hundred molecules per second -LRB- Dundr et al. 2002 -RRB- . Since proteins similarly move by diffusional motion within the cytoplasm , it stands to reason that the random scanning is also a key mechanism of protein targeting in the cytoplasm and represents a universal mechanism for how proteins find their targets . A key feature , and a requirement , in a genome-scanning model of targeting is that the interactions of proteins with chromatin are transient . This has been confirmed by photobleaching methods on a large number of DNA binding proteins -LRB- Gorski et al. 2006 ; Phair et al. 2004 ; Sprague et al. 2006 -RRB- . The transient nature of protein -- chromatin interactions is important for three reasons . First , it allows proteins to maintain a high rate of motion and thus allows faster scanning . Were protein -- chromatin interactions static , they would get stuck at non-specific or incorrect binding sites which would slow down their overall motion . Second , the short-life of protein -- chromatin interactions continuously makes available binding sites which can then be scanned by diffusing transcription factors . If proteins interacted for extended periods of time on chromatin , non-specific or improper binding would block access of the correct factors . Third , the dynamic dissociation allows for change . Were protein complexes permanently bound to their target sites , changes in transcriptional activity such as in response to physiological stimuli could only occur after active removal of the bound complex , presumably by dedicated and specialized molecular machinery . In contrast , in a dynamic binding model the natural flux of proteins provides a window of opportunity for association of a distinct regulator each time a bound protein or complex dissociates as part of its normal binding cycle . Protein dynamics as the key for formation of nuclear compartments A hallmark of the mammalian cell nucleus is the presence of distinct subnuclear compartments and domains in which particular functions occur -LRB- Handwerger and Gall 2006 ; Hernandez-Verdun 2006 -RRB- -LRB- Fig. 2 -RRB- . The prototypical nuclear compartment is the nucleolus , a distinct intranuclear compartment in which ribosomal RNAs are synthesized and partially processed . Other prominent nuclear domains include splicing factor compartments which serve as storage and assembly sites for spliceosomal components , and the Cajal bodies which are possibly involved in maturation of small nuclear RNPs . The structure of intranuclear bodies is not determined by a membrane , and the principles underlying their biogenesis are extremely poorly understood . The recently revealed dynamic properties of proteins in nuclear compartments give a hint as to the principles involved in subnuclear compartment assembly . Fig. 2Intranuclear compartments . The mammalian cell nucleus contains a larger number of distinct intranuclear compartments . The nucleolus is the site of ribosomal RNA synthesis and is a prototypical nuclear body . The complex organization of the nucleolus is revealed by multi-color staining of distinct nucleolar components . RPA43 RNA polymerase I transcription factor , Fibrillarin snRNP component , B23 rRNA processing factor . Image courtesy of Miroslav Dundr , Rosalind Franklin University of Medicine and Science , Chicago The key property in understanding the formation of nuclear compartment is the remarkable fact that the association of proteins with their compartments is highly dynamic -LRB- Dundr et al. 2004 ; Kruhlak et al. 2000 ; Phair and Misteli 2000 -RRB- . Typical residence times of proteins within a compartment are in the seconds range . In practical terms the dynamic behavior of nuclear body proteins means that they undergo repeated rapid cycles of association and dissociation between the nuclear body and the nucleoplasm . As a consequence , a nuclear body is in perpetual flux and its structure is determined by the ratio of on-rate versus off-rate of its proteins . Given these observations , the simplest scenario to explain the formation of nuclear subdomains is a self-organization model in which the collective sum of all interactions amongst proteins in a body establishes and determines its structure -LRB- Carrero et al. 2006 ; Misteli 2007 -RRB- . In this model , nuclear bodies are essentially dynamic protein aggregates which form as a consequence of multiple , transient interactions amongst a large number of proteins . This is consistent with the lack of defining intranuclear membranes and with the absence of any known dedicated structural elements within intranuclear bodies . While dynamic observations on nucleoli , splicing factor compartments and Cajal bodies support this view , this model has not been rigorously tested experimentally . A key prediction , and a way to experimentally address this fundamental question , is that it should be possible to create nuclear bodies de novo and that any nuclear body component should be able to nucleate the formation of a body . Experimental systems to test these predictions are now available , and we are eagerly awaiting the results of these key experiments . The importance of protein and RNA dynamics in regulatory events The observation of dynamic properties of proteins in the cell nucleus of living cells has suggested that dynamic trafficking is an intrinsic property of proteins and RNAs . The dynamic behavior of proteins and RNAs clearly contributes to their proper function . However , is dynamic trafficking important for physiological regulation ? Several observations demonstrate that dynamic trafficking of both proteins and RNA indeed can have regulatory function by several means . Of particular importance as a regulatory mechanism is intranuclear sequestration -LRB- Fig. 3 -RRB- . Accumulation of a protein at a particular nuclear site or within a nuclear compartment can serve to either increase the local concentration of a factor at that site or to reduce its abundance in the rest of the nucleus . Several proteins are now known to accumulate , in particular nuclear locations in response to physiological cues and their sequestration is thought to contribute to their cellular function . Fig. 3Intranuclear retention and sequestration as a regulatory means . a Retention of proteins within intranuclear subcompartments regulates their nuclear concentration and their rate of nuclear export . b Sequestration of proteins at the nuclear periphery , particularly via interaction with the lamina -LRB- blue -RRB- controls their nucleoplasmic concentration and their availability at target genes . Sequestration can act both as a negative or a positive regulatory mechanism . Sequestration of an activator leads to repression , sequestration of a repression leads to activation . c Retention of partially or fully processed RNA within intranuclear compartments modulates RNA export rate Prototypical example of sequestration as a regulatory mechanism is the accumulation of the ubiquitin ligases MDM2 and the von Hippel-Lindau tumor suppressor protein VHL in the nucleolus -LRB- Mekhail et al. 2005 , 2007 -RRB- . These two proteins are responsible for the ubiquitination of the p53 tumor suppressor and the hypoxia-induced factor HIF , respectively . Modification of these two targets leads to their nuclear export and degradation in the cytoplasm . Since ubiquitination is a major regulatory mechanism in their function , the concentration of the ubiquitin ligase in the nucleus significantly determines their fate . Both MDM2 and VHL diffuse freely within the nucleus . However , in response to physiological cues , both MDM2 and VHL can become sequestered in the nucleolus , thus reducing their nucleoplasmic concentration and the extent of ubiquitination of their targets -LRB- Mekhail et al. 2005 , 2007 -RRB- -LRB- Fig. 1a -RRB- . Sequestration of these proteins appears to occur by increased retention of diffusing molecules within the nucleolus . How this retention is regulated is currently unclear -LRB- Mekhail et al. 2005 , 2007 -RRB- . The possibility that retention is a highly controlled mechanism comes from observations on nucleostemin , a nucleolar protein implicated in stem cell maintenance and cancer proliferation -LRB- Meng et al. 2007 ; Tsai and McKay 2005 -RRB- . The retention of nucleostemin in the nucleolus is controlled by a GTP -- GDP binding cycle which might be directly or indirectly linked to signaling pathways . In addition to nucleostemin the localization of several additional GTP-binding domain containing nucleolar proteins seems to be controlled in a similar fashion -LRB- Meng et al. 2007 ; Tsai and McKay 2005 -RRB- . Intranuclear sequestration can also be a means of negative regulation . The basic helix -- loop -- helix transcription factor Hand1 is expressed in trophoblast stem cells and is required for their differentiation along several lineages -LRB- Martindill et al. 2007 -RRB- . Hand1 is sequestered in the nucleolus and upon stimulation during differentiation is rapidly released and moves into the nucleoplasm where it presumably acts on target genes . Hand1 is retained in the nucleolus by its interaction with I-mfa , and this interaction is sensitive to phosphorylation of Hand1 . Upon phosphorylation Hand1 dissociates from I-mfa and is released from the nucleolus . Interestingly , both the sequestration of Hand1 in the nucleolus and its release are functionally important . Premature release of Hand1 triggers differentiation , whereas the presence of Hand1 is also required to maintain the stem-cell potential of trophoblast stem cells . Thus nucleolar sequestration of this key cell fate regulator serves a dual regulatory role -LRB- Martindill et al. 2007 -RRB- . Most examples of intranuclear sequestration involve the nucleolus . However , the nuclear lamina has recently also emerged as a major site for transcription factor sequestration -LRB- Heessen and Fornerod 2007 -RRB- -LRB- Fig. 1b -RRB- . Prominent transcription factors which associate with the nuclear periphery either via interaction with the lamins or with the inner nuclear membrane proteins include c-fos , Oct-1 and Rb -LRB- Imai et al. 1997 ; Ivorra et al. 2006 ; Johnson et al. 2004 -RRB- . It is tempting to speculate that the association of these factors with the periphery reduces the intranuclear concentration and thus their availability at target genes -LRB- Heessen and Fornerod 2007 -RRB- . A physiological role for such peripheral sequestration is most strongly suggested by observations on the pro-proliferation factor c-fos which associates with the lamina in quiescent cells but is released upon entry of cells into the cell cycle correlating with activation of some of its target genes -LRB- Ivorra et al. 2006 -RRB- . Similarly , release of Oct-1 from the lamina correlates with activation of collagenase , one of its prime target genes -LRB- Imai et al. 1997 -RRB- . The sequestration of transcription factors at the nuclear periphery is a potentially powerful and simple mechanism of transcriptional regulation . It will be important to determine how widespread this mechanism is and how precisely the association of transcription factors with the lamina is controlled . Dynamic trafficking of RNA is similarly used as a regulatory mechanism -LRB- Prasanth and Spector 2007 -RRB- . In particular , it appears that RNA retention can act as a quality control mechanism for RNA export . For one , incompletely spliced or processed RNAs are retained at their sites of transcription and not released into the nucleoplasm for export . In addition , pools of stable polyadenylated RNAs are enriched in the nuclear splicing factor compartments , although their function remains unclear -LRB- Huang et al. 1994 -RRB- . But retention is also emerging as a mechanism for regulation of specific RNAs . A non-coding RNA transcribed from the mouse Cat2 gene encoding a cationic amino acid transporter is retained in the nucleus via a dedicated 3 ′ end containing extensive repeat sequences -LRB- Prasanth et al. 2005 -RRB- . These sequences are responsible for accumulation of the RNA in a nuclear compartment identified as paraspeckles -LRB- Fig. 1c -RRB- . This retention serves a physiological function because upon stress the Cat2 RNA is cleaved , released from paraspeckles and rapidly exported into the cytoplasm for translation , thus ensuring a rapid physiological stress response . Although the Cat2 RNA is to-date the best characterized example of such retention , a similar mechanism is likely at work for the migration-stimulating factor -LRB- MSF -RRB- mRNA whose 3 ′ end resembles that of Cat2 and is retained within the nucleus -LRB- Kay et al. 2005 -RRB- . Under conditions of MSF secretion the RNA appears to be released from the nucleus and rapidly translated . A further candidate for control via nuclear retention is the neuron-specific gomafu RNA which is retained in intranuclear compartments . Interestingly , gomafu does not seem to encode for any protein and might thus be a non-coding regulatory RNA which is retained in the nucleus -LRB- Sone et al. 2007 -RRB- . Conclusions The past few years have seen a dramatic change in how we view the cell nucleus . We have come to appreciate the presence of distinct structural elements within the nucleus , the presence of a multitude of intranuclear bodies and the fact that genomes are non-randomly organized within the nuclear space . One of the most consequential findings has been the realization that just about every aspect of nuclear organization is highly dynamic . Both proteins and RNAs move rapidly within the nucleus , and they only transiently interact with chromatin and nuclear bodies . It is now clear that the dynamic nature of nuclear components is a fundamental property and has implications for how molecules are targeted to their final destinations and for how intranuclear compartments form . Most importantly , it is becoming clear that the dynamic properties of nuclear proteins are critical for various mechanisms of physiological regulation , particularly via sequestration and retention of proteins and RNA . It is likely that these recently discovered examples of regulation by modulation of dynamic interactions within the cell nucleus are only the proverbial tip of the iceberg , and it stands to reason that we should consider the contribution of dynamic interactions and trafficking in any nuclear event we investigate in the future .

O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

nuclear architecture
dynamics
diffusion
rna