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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Keywords:Copyright 2011 by ESPGHAN and NASPGHAN
Crohn disease; inflammation; microarray; transcription factors; ulcerative colitis