Transglutaminase 2 expression is enhanced synergistically by interferon-γ and tumour necrosis factor-α in human small intestine

Abstract

Transglutaminase 2 (TG2) is expressed ubiquitously, has multiple physiological functions and has also been associated with inflammatory diseases, neurodegenerative disorders, autoimmunity and cancer. In particular, TG2 is expressed in small intestine mucosa where it is up-regulated in active coeliac disease (CD). The aim of this work was to investigate the induction of TG2 expression by proinflammatory cytokines [interleukin (IL)-1, IL-6, tumour necrosis factor (TNF)-α, interferon (IFN)-γ and IL-15] and the signalling pathways involved, in human epithelial and monocytic cells and in intestinal tissue from controls and untreated CD patients. Here we report that IFN-γ was the most potent inducer of TG2 expression in the small intestinal mucosa and in four [Caco-2, HT-29, Calu-6 and human acute monocytic leukaemia cell line (THP-1)] of five cell lines tested. The combination of TNF-α and IFN-γ produced a strong synergistic effect. The use of selective inhibitors of signalling pathways revealed that induction of TG2 by IFN-γ was mediated by phosphoinositide 3-kinase (PI3K), while c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) were required for TNF-α activation. Quantitative polymerase chain reaction (PCR), flow cytometry and Western blot analysis showed that TG2 expression was blocked completely when stimulation by either TNF-α or IFN-γ was performed in the presence of nuclear factor (NF)-κB inhibitors (sulphasalazine and BAY-117082). TG2 was up-regulated substantially by TNF-α and IFN-γ in intestinal mucosa in untreated CD compared with controls. This study shows that IFN-γ, a dominant cytokine in intestinal mucosa in active CD, is the most potent inducer of TG2, and synergism with TNF-α may contribute to exacerbate the pathogenic mechanism of CD. Selective inhibition of signalling pathways may be of therapeutic benefit.

Fig. 1

Proinflammatory cytokines regulate transglutaminase 2 (TG2) expression in human cell lines. Expression of TG2 was determined by quantitative real time polymerase chain reaction (RT–PCR) in five different human cell lines: intestinal epithelial cell lines Caco-2 and HT29, lung epithelial cell lines A549 and Calu-6 and the monocytic cell line human acute monocytic leukaemia cell line (THP-1) (lower panel of the chart) stimulated for 24 h with proinflammatory cytokines: tumour necrosis factor (TNF)-α (10 ng/ml), interferon (IFN)-γ (200 UI/ml), interleukin (IL)-1 (10 ng/ml), IL-6 (10 ng/ml) and IL-15 (20 ng/ml). Results were normalized against β-actin and relative TG2 mRNA levels were referred to the non-stimulated control (value = 1). Data represent means ± standard error of the mean (n = 3). The Mann–Whitney U-test was performed: *P < 0·05; **P < 0·01.

Fig. 2

Tumour necrosis factor (TNF)-α and interferon (IFN)-γ synergistically induce transglutaminase 2 (TG2) expression. Expression of TG2 was determined by quantitative real time polymerase chain reaction (RT–PCR) in Caco-2, HT29, A549, Calu-6 and THP-1 cells (lower panel of the chart) stimulated for 24 h with TNF-α (10 ng/ml), IFN-γ (200 UI/ml) or a combination of both. Results were normalized against β-actin and relative TG2 mRNA levels were referred to the non-stimulated control (value = 1). Data represent means ± standard error of the mean (n = 3). The Mann–Whitney U-test was performed: *P < 0·05; **P < 0·01.

Fig. 3

Effect of inhibitors of signalling pathways on transglutaminase 2 (TG2) induction by tumour necrosis factor (TNF)-α and interferon (IFN)-γ. Caco-2 and THP-1 cells were incubated for 24 h with specific inhibitors of signalling pathways: SP600125 (20 µM), SB203580 (10 µM), wortmannin (10 µM), Ly294002 (2 µM), sulphasalazine (10 µM) in the presence of TNF-α (10 ng/ml), IFN-γ (200 UI/ml) or the double stimulus TNF-α + IFN-γ. Level of TG2 transcripts was determined by quantitative real time polymerase chain reaction (RT–PCR). Results were normalized with the housekeeping gene β-actin and were referred to the non-stimulated control (value = 1). Data represent means ± standard error of the mean (n = 4). The Mann–Whitney U-test was performed: *P < 0·05; **P < 0·01 (asterisks on individual bars indicate comparison versus control). The inserted chart indicates the signalling pathways targeted by the inhibitors used.

Fig. 4

Tumour necrosis factor (TNF)-α and interferon (IFN)-γ enhance transglutaminase 2 (TG2) promoter activity. Caco-2 cells were transfected transiently with the pTG2–luciferase plasmid. Luciferase activity (Dual LR System; Promega) was determined after 24 h of stimulation with TNF-α (TNF-α 10 ng/ml), IFN-γ (200 UI/ml) or TNF-α + IFN-γ alone or in the presence of sulphasalazine (10 µM), Ly294002 (2 µM) or SP600125 (20 µM). Relative luciferase units (RLU) are referred to the non-stimulated control. Data represent means ± standard error of the mean (n = 3). The Mann–Whitney U-test was performed: *P < 0·05 (asterisks on individual bars indicate comparison versus control).

Fig. 5

Tumour necrosis factor (TNF)-α and interferon (IFN)-γ induce transglutaminase 2 (TG2) synthesis. TG2 expression was analysed by Western blot on protein extracts obtained from Caco-2 cells stimulated 20 h with TNF-α (10 ng/ml) and IFN-γ (200 UI/ml) (lane 2), medium (control, lane 3), (TNF-α + IFN-γ) + sulphasalazine (10 µM) (lane 4) and (TNF-α + IFN-γ) + wortmannin (10 µM) (lane 5). Anti-TG2 4E1G9 monoclonal antibody was used for TG2 detection. Detection of β-actin (Santa Cruz Biotechnology) was used as normalizer. Recombinant TG2 (rTG2) (lane 1) produced in our laboratory was used as a reference.

Fig. 6

Tumour necrosis factor (TNF)-α and interferon (IFN)-γ induce surface expression of transglutaminase 2 (TG2). Surface TG2 expression was evaluated by flow cytometry on non-stimulated THP-1 cells (grey line) or in cells stimulated for 20 h with TNF-α (10 ng/ml) and IFN-γ (200 UI/ml) either in the absence (black line) or the presence of sulphasalazine (10 µM) (dashed line). Cells were labelled with the 1H7H9 anti-TG2 monoclonal antibody or an isotype control (grey histogram). Mean fluorescence intensity (MFI) values are indicated for each histogram.

Fig. 7

Transglutaminase 2 (TG2) expression in human small intestinal mucosa. Duodenal samples from untreated coeliac disease (CD) patients (n = 5) and control individuals (n = 7) were incubated for 24 h with tumour necrosis factor (TNF)-α (10 ng/ml) and interferon (IFN)-γ (200 UI/ml) or medium (NS) (a) or with TNF-α + IFN-γ in the presence of sulphasalazine (10 µM) or Ly294002 (2 µM) (b). In (a) the levels of TG2 transcript were determined by quantitative real time polymerase chain reaction (RT–PCR) as fold increase (ΔCt) in comparison with the housekeeping gene (β-actin). In (b), the fold increase (2⁁Δ^Ct) of the TG2 transcript was normalized with the housekeeping gene (β-actin) and referred to the unstimulated tissue from each patient. Mann–Whitney U-test: *P < 0·05; **P < 0·01.

Fig. 8

Model of transglutaminase 2 (TG2) transcriptional activation by tumour necrosis factor (TNF)-α and interferon (IFN)-γ. Signalling pathways and transcription factors activated by TNF-α and IFN-γ that regulate TG2 transcription. Role of TG2 in different chronic disorders.