Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn disease (CD), are chronic, spontaneously relapsing, immunologically mediated disorders of the gut. The current understanding of IBD pathogenesis suggests a complex interplay of multiple environmental and genetic factors that results in a dysregulation of the host immune response, with an inappropriate activation of the intestinal mucosal immune system leading to inflammation and tissue damage (1–3). The inflamed tissue in patients with IBD with active disease is characterized by an increased production of proinflammatory cytokines, which represent the principal target of treatment strategies (4). Because most cytokines exert their biological properties through a downstream pathway that involves activation of corresponding signal transducers and activators of transcription, recent research has focused on these signaling pathways (5). However, the pivotal elements in the regulation of the inflammatory response remain unclear and additional studies are necessary.
One of the transcription factors involved in the pathogenesis of IBD belongs to the NF-κB family, whose components regulate multiple inflammatory target genes, including several NF-κB genes themselves, cytokines and chemokines, antiapoptotic and proliferative genes, and adhesion molecules (6,7). In a previous study (8), we demonstrated that NF-κB is strongly activated in inflamed tissues of pediatric patients with CD as compared to healthy controls.
Recently, the study of the disease mechanisms in pediatric IBD has raised great interest among scientists and clinicians. A growing view is that IBD in children represents a unique population in which the disease is in an early stage and the mechanisms are poorly confounded or influenced by environmental factors. One of the most challenging issues in pediatric IBD is how to translate advancements in the knowledge of pathogenesis into therapeutic strategies aimed at slowing down, if not preventing, the progression into a late disease stage (9).
The present study is aimed at identifying, through a microarray assay, new transcription factors involved in the pathogenesis of IBD and at establishing among them early biomarkers of inflammation and potential targets for therapeutic intervention. We found that several genes involved in fundamental signaling of inflammatory and/or immune response were mostly overexpressed in patients with IBD as compared with controls.
PATIENTS AND METHODS
All of the patients were enrolled at the Pediatric Gastroenterology and Liver Unit of the Sapienza University of Rome and underwent ileocolonoscopy performed by the same endoscopist with a pediatric video colonoscope (Olympus PCF 40L Olympus, Tokyo, Japan) after conscious sedation with intravenous pethidine (1–2 mg/kg) and midazolam (0.1 mg/kg) or general anesthesia.
Diagnosis of CD and UC was based on widely agreed-upon endoscopic and histological criteria as well as on the exclusion of infectious and systemic disease, food allergies, and malabsorption syndromes (10). Clinical disease activity was measured using established clinical parameters of the pediatric CD and UC activity index (11,12). Tables 1 and 2 (12,13) report the demographic and clinical characteristics of the patients.
Three treatment-naïve pediatric patients with active colonic CD (1 girl, mean age 14.7 years) were recruited for polymerase chain reaction (PCR) microarray experiments. These patients showed similar degrees of disease activity and histological inflammation. The biopsies were taken from inflamed and uninflamed areas of the colon. Three normal subjects (1 girl, mean age 13.2 years) with functional gastrointestinal disorders and with normal colonoscopy and histology served as controls.
To validate microarray results, 28 patients with CD, including the previous 3 (9 girls, mean age 14.3 years, range 8–18 years), and 15 patients with UC (6 girls, mean age 7.9 years, range 1–17 years) were analyzed by real-time PCR (RT-PCR). In these patients, the biopsies were taken from both involved and uninvolved tissues.
When included in the study, 9 patients with CD and 3 patients with UC were not receiving any treatment, whereas the others were treated with different drugs including immunomodulators (azathioprine), mesalazine, or oral corticosteroids at low doses; some patients with CD were receiving courses of nutritional therapy. Tables 1 and 2 also summarize the therapy at time of endoscopy. The control population consisted of 20 children, including the previous 3 (11 girls, mean age 13.5 years, range 9–17), investigated for symptoms and signs of functional gastrointestinal disorders, without organic or inflammatory disease, as documented by normal endoscopy and histology.
Informed Consent and Approval by the Ethics Committee
The study was approved by the ethics committee of the University Hospital Umberto I where patients were admitted. For each patient informed consent from parents was obtained.
Mucosal biopsy specimens, taken from ileal and colonic districts, were conserved in RNAlater (Ambion) before RNA extraction; samples for protein analysis were immediately snap-frozen in liquid nitrogen.
Target Preparation and PCR Arrays
Total RNA was isolated using the RNeasy Kit (QiaGen GmbH, Hilden, Germany). RNA was quantified using the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE) and its integrity was checked by gel electrophoresis. Of the total RNA pooled from 3 selected patients with CD and 3 controls, 1.5 μg was reverse transcribed to complementary DNA (cDNA) by RT First Strand Kit (SA Biosciences, Frederick, MD). Diluted cDNA from each sample was mixed with 2x SuperArray RT qPCR Master Mix. Each well of the 96-well PCR array (SABiosciences, cat PAHS-075) was loaded with 25 μL of Reaction Mix. A 2-step cycling program was performed: 1 cycle of 10 min at 95°C followed by 40 cycles of a denaturation step at 95°C for 15 sec and 1 min at 60°C. Results were normalized using 2 housekeeping genes: β-actin and glyceraldehyde-3-phosphate dehydrogenase.
PCR array Data Analysis Web Portal automatically performs calculations and interpretations of control wells upon including threshold cycle data from a real-time instrument. ΔΔCt method was used for gene expression determination. Appropriate controls of DNA contamination, reverse transcription, and PCR efficiency were performed.
PCR Arrays Reliability and Reproducibility
To ascertain data reliability and reproducibility, the PCR array experiments were performed on triplicate using the same pool of target RNA.
Independent Quantitation of Microarray Results by RT-PCR
Selected gene expression signals were independently quantitated with RT-PCR on patients with CD and UC. Primers were designed to nonredundant sequences using Primer Express V3.0 (Applied Biosystems, Foster City, CA). Total RNA (1 μg) was reverse transcribed to cDNA by a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). RT-PCR amplification was done with an ABI PRISM 7300 Sequence Detection System, using the SYBR Green kit (Applied Biosystems). The primers used are listed in Table 3. Relative transcript levels were determined using β-actin as the endogenous control gene.
Snap-frozen biopsy specimens were homogenized in ice-cold lysis buffer and the protein concentration was determined by the Bradford assay (Bio-Rad Laboratories, Hercules, CA). Equal amounts of proteins were loaded in each lane and run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Proteins were transferred to a polyvinylidene difluoride membrane (Amersham, Little Chalfont, UK). Anti-human activating transcription factor 3 (ATF3) and anti-human hypoxia-inducible transcription factor-1α (HIF1α) polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were used as primary antibodies. Goat anti-rabbit antibody conjugated to horseradish peroxidase (Santa Cruz) was used as a secondary antibody. Specific signals were detected using the ECL reagents (Amersham, Biosciences Europe GmbH, Freiburg, Germany) for chemiluminescence. The anti-β-actin monoclonal antibody (Sigma, St Louis, MO) was used to control the equivalence of protein loading for total extracts. Densitometrical analysis of the blots was performed by a GS-700 densitometer (Bio-Rad) using the Software Quantity One (Bio-Rad).
Cell Line and Culture Conditions
The human colon adenocarcinoma cell line Caco2 was obtained from American Type Culture Collection (Rockville, MD). Caco2 cells were grown in Eagle's minimum essential medium (Sigma, St Louis, MO) and supplemented with 10% fetal bovine serum, 2 mmol/L L-glutamine, 1% nonessential amino acids, and 1% penicillin/streptomycin. Cytokine inductions were performed by treating cells at 70% of confluence with cytomix (10 ng/mL of tumor necrosis factor-α and 1000 U/mL of interferon [INF]-γ, Peprotech, Inalco, Milan, Italy) for different times. Hypoxia was induced by adding to medium 200 μmol/L of cobalt chloride (Sigma). Dimethylthiazol-diphenyltetrazolium bromide (MTT) assay was used to check cell viability after treatments, according to manufacturer instructions (Sigma).
All of the experiments on tissues and cell lines were repeated 3 times. Statistically significant differences between control and IBD samples were determined using a Mann-Whitney U test. A P value <0.05 was considered significant.
Expression Profiles of the CD Colonic Mucosa
The messenger RNA (mRNA) pooled from colonic tissue samples of the CD1, CD2, and CD3 patients and 3 healthy controls was used for gene expression profiling. We used RNA from whole colonic mucosa, which comprises heterogeneous cell types, aiming at gaining a global and representative insight into cellular changes associated with CD pathogenesis. Both uninflamed and inflamed colonic mucosa samples were collected from patients and controls. The array includes 84 transcription factors downstream of signaling from cytokines and chemokines; growth factors such as BMP, EGF, IGF, insulin, PDGF, TGF-β, TPO, and VEGF; and signaling from androgen, B-cell, G-protein-coupled, T-cell, and Toll-like receptors. The array also includes target transcription factors in signal transduction pathways such as JAK/STAT, JNK, and other MAP kinases; NF-κB; Notch; and WNT. A 2.5-fold cutoff was applied to identify genes that were consistently upregulated in patients with CD as compared with healthy controls. At this cutoff level, 40 genes were found to be significantly upregulated (P < 0.01) between inflamed colonic mucosa of patients with CD and controls (Fig. 1A). Of these 40 genes, 17 transcripts were also identified as differentially regulated at a significant level (P < 0.01) between uninflamed colonic mucosa of patients with CD and normal controls (Fig. 1B). STAT1 and ATF3 were the most upregulated genes in inflamed tissue (-fold change = 13.7 and 13.0, respectively). STAT1 was also the most activated transcription factor in uninvolved mucosa (-fold change = 7.2), whereas ATF3 did not show changes in these areas. The differential expression and the functional groups of all overactivated transcription factors are shown in Table 4.
RT-PCR Validation of Selected Transcripts
RT-PCR on 10 selected transcripts was used for the validation of expression microarray results. Genes were selected on the basis of their high expression level (-fold change ≥5) and their unknown role in CD: STAT1, ATF3, SMAD9, IFN-regulatory factor-1 (IRF1), HIF1α, C/EBPβ, ETS2, E2F6, FOXA2, and JUND. The mRNA expression analysis was performed on either colonic and ileal districts in involved and uninvolved areas, and the population of 3 subjects used for microarray studies was increased to 28 patients with CD (Table 1), 15 patients with UC (Table 2), and 20 controls. Although some expected individual variability was observed, all 10 tested transcription factors were found significantly upregulated (P < 0.05) in the inflamed colonic and ileal mucosa of patients with CD as compared with healthy controls (Fig. 2A). No statistical differences between colonic and ileal expression level for these selected transcription factors were found. Four of them (STAT1, HIF1α, IRF1, and SMAD9) resulted to be upregulated (P < 0.05) also in the uninflamed mucosa of patients with CD (Fig. 2B). Due to the low number of biopsies taken from patients, uninflamed tissue includes either ileal or colonic districts. These data were in agreement with the microarrays results. The same 10 transcription factors were also analyzed in patients with UC; 4 of them (ATF3, HIF1α, FOXA2, and STAT1) were significantly upregulated in the inflamed, whereas only 2, HIF1α and STAT1, in the uninflamed colon (Fig. 2C, D) were, as compared with healthy controls.
ATF3 and HIF1α Protein Expression in Mucosal Biopsies
Two overexpressed genes were selected for their emerging role in inflammatory response and for their recently reported functional interaction with NF-κB, the master regulator of immune response: ATF3 (14) and HIF1α (15). To assess whether a protein overexpression was following their transcriptional upregulation, Western blot assay was performed in ileal and colonic protein extracts of patients with CD and controls. Protein levels of both genes also exhibited significant increases (P < 0.05) in affected ileal and colonic mucosa of patients with CD as compared with controls, whereas in unaffected mucosa only HIF1α were significant upregulated, thus confirming the results of RT-PCR analysis (P < 0.05) (Fig. 3).
ATF3 and HIF1α Protein Expression in Caco2 Cell Line
To assess whether these 2 genes responded to inflammatory stimuli, the human cell line Caco2, a model of the intestinal barrier, was treated with cytomix (TNF-α and INF-γ), and in a separate experiment, with cobalt chloride (CoCl2) to induce hypoxia. A combined treatment with cytokines and CoCl2 was also performed. ATF3 and HIF1α protein expression were detected after 3, 6, and 24 hours of treatment by Western blot experiments. After cytokines induction, ATF3 protein level significantly increased after 3 hours and remained at the same level for 24 hours, whereas HIF1α increases significantly only after 24 hours (Fig. 4A). In hypoxic conditions, ATF3 showed a maximum increase after 24 hours (3.8-fold), whereas HIF1α steeply increased after 3 hours of treatment, with a maximum after 6 hours (6.7-fold) (Fig. 4B). Combined treatment with cytokines and CoCl2 had an additive effect on expression of both genes, but at different time points: HIF1α presented a maximum increase after 3 hours of treatment (6.2-fold), and then rapidly decreased, whereas ATF3 progressively increased up to 5.6-fold, after 24 hours (Fig. 4C). Increased levels were considered significant when P < 0.05. No significant decrease in cell viability, measured by MTT assay, was detected after 24 hours of treatment with cytomix or hypoxia (viability 100% and 95%, respectively), whereas a slight decrease of viability (viability 82%) was found only after the combined treatment.
The objective of this investigation was to analyze a large repertoire of transcription factors and elucidate all differentially regulated factors in the mucosal biopsies of pediatric patients with CD, using PCR microarrays. With this approach we aimed at identifying new expression patterns associated with IBD and eventually to individuate novel markers of inflammation and candidates for gene therapy. It is worth noting that there are relatively few studies, especially in pediatrics, using microarrays technology that is directly applied to IBD biopsies (16–19).
It is remarkable that most of the transcription factors analyzed by microarrays were upregulated in involved colonic areas; interestingly, a subset of them was also upregulated in uninvolved colonic areas of the patients. It was particularly attractive to select 10 genes from those showing the highest levels of mRNA expression and perform a more detailed analysis by quantitative RT-PCR; for this purpose more subjects were recruited, including patients with UC, and other intestinal districts such as ileum were analyzed. At microarray, STAT1 was the most upregulated gene both in inflamed and in uninflamed tissue (-fold change of 13.8 and 7.2, respectively) as compared with controls. This was not unexpected because STAT1 is a component of the activators of transcription family (STAT) playing a critical role in the transcriptional response to cytokines, albeit the specific role of each member of the family remains somewhat unclear (20). STAT1 seems to be specifically involved in INFs signaling also by an interaction with IRF1, a gene involved in innate and adaptive immune responses and overactivated in our experiments in patients with CD (21).
ATF3, SMAD9, and HIF1α were also markedly upregulated in inflamed colonic and ileal mucosa of patients with CD as compared with controls; SMAD9 and HIF1α were also active in uninflamed tissue. These genes are usually involved in immune reactions, but their role in IBD has still to be clarified. ATF3 is able to respond to a variety of stress signals (14), SMAD9 belongs to a family of regulators of TGF-β signaling (22), whereas HIF1α is a subunit of HIF, a principal regulator of cell response to hypoxia (15).
There are other transcription factors with a known role in immunity; however, their involvement in IBD has not been explored. C/EBP-β is an important regulator of genes involved in immune and inflammatory responses and has been shown to regulate IL-6 production in human enterocytes (23). FOXA2 is involved in goblet cell differentiation and in regulation of intestinal epithelial mucin expression (24). ETS2, a member of a family of 29 transcription factors activated by proinflammatory cytokines, growth factors, and vasoactive peptides, is overexpressed in a number of inflammatory/autoimmune diseases (25). JUND is a member of the Jun family of proteins, which are primary components of the activator protein 1 (AP-1) transcription factor: it plays a critical role in maintaining epithelial barrier function (26), is a negative regulator of T-cell activation, and controls cytokines expression (27). E2F6 is a potent transcriptional repressor playing important roles in cell cycle regulation and proliferation; however, its role in immune/inflammatory diseases is still unknown (28). In patients with UC, a distinctive gene expression pattern was obtained: in the inflamed colon 4 transcription factors (ATF3, HIF1α, FOXA2, and STAT1) were upregulated and 2 of these (HIF1α and STAT1) were activated in the uninflamed colon. Surprisingly, transcription factors SMAD9, C/EBPβ, and IRF1, strongly activated in CD, did not vary in UC, suggesting that different molecular mechanisms underlie the pathogenesis of these 2 entities and that subsets of genes differentially expressed could be used in the future as distinctive markers of CD or UC.
An interesting aspect of this study was the significant increase in the expression of several genes in the unaffected mucosa of patients with CD as compared with controls. The most expressed transcription factors in these tissues were STAT1, HIF1α, IRF1, and SMAD9. This suggests that intestinal inflammation in CD, despite absence of obvious endoscopic and histological alterations, can be activated at the molecular level. This also suggests involvement of these genes in the early phases of the disease, thus investigating their signaling pathways could be a promising target for innovative therapeutic interventions.
Recently, inflammation has been described as a multicomponent response to tissue stress, injury, and infection, consisting of sequential activation of multiple gene sets or transcriptional modules that are coordinately regulated by dedicate transcription factors (29). Some factors belong to the primary response gene group, such as NF-κB and IRF1, whereas others, such as ATF3 and C/EBP-β, appear to be activated in a secondary response. This could explain the differential expression of transcription factors in affected and unaffected mucosal areas as compared with controls. Indeed, IRF1, SMAD9, STAT1, and HIF1α, which were activated both in involved and in uninvolved CD tissues, could be thought to belong to the primary response and suggested as early markers of the disease; ATF3 and C/EBP-β, which are activated only in CD inflamed tissues, could be involved in a secondary response of the inflammatory process and considered specific markers of inflammation.
In the second part of the study, our interest was focused on 2 transcription factors, recently found to be involved in immunity and functionally related to NF-κB, the master regulatory factor of inflammatory response (30–32): ATF3 and HIF1α. They were markedly overexpressed in the inflamed intestinal mucosa of children with active IBD. HIF1α was activated also in uninvolved areas of the intestine. This upregulation involves both mRNA and protein expression, indicating that their regulation was mainly at the transcriptional level. The present study is the first demonstration of the strong involvement of these 2 transcription factors in the pathogenesis of IBD and could represent a stimulus to further elucidate their role.
ATF3 is a member of the CREB family of basic leucin zipper transcription factors with a still obscure biological role: it has been shown as a transcriptional activator or repressor, depending on the cell type and stimulus (14). ATF3 expression is maintained at low levels in quiescent cells but is greatly induced by a variety of stress signals in vivo and in vitro (33). This transcription factor has recently been identified as a potent negative regulator of the inflammatory response in macrophages, where it antagonizes NF-κB-induced responses (34,35). The role of ATF3 in immune responses has only recently been described; indeed, ATF3 is able to negatively regulate transcription of lipopolysaccharide-induced proinflammatory cytokines such as IL-6 and IL-12 (34). Our results show for the first time the involvement of ATF3 in the inflammatory process of IBD, although its exact role remains to be clarified.
HIF1α is a subunit of HIF, the main transcription factor activated by hypoxia. Inflamed intestinal mucosa is characterized by a reduced oxygen levels when compared with healthy mucosa (36,37). An interdependency between HIF1α and NF-κB in inflammation has recently been demonstrated (30): activation of the NF-κB pathway leads to transcriptional upregulation of HIF1 mRNA expression, and hypoxia, by modulation of IκB kinase, activates NF-κB signaling (31). Few and controversial studies have explored the role of HIF1α in the IBD mechanisms. A protective role of HIF1α has been reported in murine experimental colitis (38,39); however, a study of Shah et al (40) demonstrated that a chronic increase in HIF signaling in murine colon epithelial cells worsens disease progression. In our study, HIF1α is strongly activated both in inflamed and uninflamed tissue of CD and UC, suggesting a role for this gene in the early phases of disease.
ATF3 and HIF1α are also known to be activated by TGF-β, a pluripotent cytokine that regulates epithelial tissue homeostasis and, by increasing DNA binding activity of HIF1α, upregulates vascular endothelial growth factor (41), suggesting a role for these genes in an enhanced regulatory immune response and in angiogenesis, a condition of chronic inflamed mucosa.
To support involvement of the 2 genes in the inflammatory process, in vitro experiments were performed on Caco2, a cell line representing a model of intestinal epithelium. Both genes responded to proinflammatory stimuli as cytokines and hypoxia and the response appeared to be directly proportional to the intensity of stimuli. Indeed, an additive effect of the combined treatment was shown, but at different time points: ATF3 presented a late response compared with HIF1α. Although additional studies are needed, in particular to elucidate the cross-talk between these 2 genes and NF-κB signaling, we propose ATF3 and HIF1α as novel candidates involved in the pathogenesis of IBD.
In conclusion, the present study shows for the first time to the authors’ knowledge overactivation of most transcription factors in inflamed and uninflamed intestinal mucosa of pediatric patients with IBD. These results can open new perspectives in the knowledge of disease mechanisms, leading to the identification of specific markers of inflammation and therapeutic targets.
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