Functional analysis of Transglutaminase 2 promoter

BAYARDO; CHIRDO F

Amsterdam

Congreso; 13th International Coeliac Disease Symposium; 2009

International Coeliac Disease Symposium

Transglutaminase 2 (TG2) was identified as the main autoantigen detected in the anti-endomisium determination, test used in coeliac disease (CD) screening. Although the pathogenic role for anti-TG2 antibodies has not been definitely proven, detection of anti-TG2 specific antibodies in serum is nowadays commonly used in serological assays for CD detection.        Besides, TG2 is directly involved in CD pathogenesis. Selective deamidation of glutamine residues in gliadin derived peptides leads to a higher affinity binding to the HLA predisposing alleles DQ2 (A1*0501, B1*0201) and DQ8 (A1*0301, B1*0301), and also to a higher gliadin specific T cell stimulation. The biology of TG2 in the intestinal mucosa is still poorly understood. The main cellular source as well as its regulation is unknown. TG2 is strongly upregulated in villus atrophy, the histological hallmark of CD.The aim of this work was to do a functional analysis of TG2 promoter by studying the effect of different stimulus on the TG2 expression in two human intestinal epithelial cell lines (Caco-2 cells, the most commonly used in vitro model for enterocytes, and HT29 cells) and in two human pulmonary epithelial cell lines (A549 and Calu-6).We have recently cloned the TG2 gene from Caco-2 cells (Genbank AY675221). We used the reported sequence to design a specific pair of primers to amplify a 243 bp tTG fragment. Real Time PCR was performed to determine the TG2 RNAm level after different treatments.In order to analyze specific transcriptional activity of TG2 promoter, we cloned a region of 1.5kb upstream of firefly luciferase reporter gene, and we transfected the epithelial cell lines with this construction. Luciferase activity was measured after different treatments. Preliminary results shown that simultaneous incubation for 24h of TNFa and IFNg produces the higher increase in tTG mRNA levels by Real Time PCR (30.48 2^Ct in A549, 38.18 2^Ct in Calu-6, 45 2^Ct in Caco-2 and 55.71 in HT29), and luciferase activity, in all four cell lines.IL-15 and IL-1 upregulated tTG in A549 but not in Caco-2, while L-6 stimulated only in HT29 but not in the other cell lines.Different ligands of innate immunity, such as poli I:C (TLR3) and flagellin (TLR5), shown to upregulate tTG only in the pulmonary epithelial cell lines (A549 and Calu-6). Since TNFa can modulate tTG expression through the NFkB pathway, in liver cells (Kuncio et al., 1998), and tTG can directly activate NFkB pathway leading to a complex regulation mechanism (Lee et al., 2004), we decided to evaluate the effect of adding an NFkB inhibitor (Sulfasalazine) in our different stimulations. Interestingly, we found that the induction of tTG mRNA levels due to simultaneous incubation of TNFa and IFNg was partially inhibited when 2 h prior to this stimuli we added the NFkB inhibitor. We found the same result in all the cell lines studied. The inhibitory effect of sulfasalazine was even higher when the NFkB inhibitor was added in simultaneous with the mix of TNFa and IFNg.