Ordered transcriptional factor recruitment and epigenetic regulation of tnf-alpha in necrotizing acute pancreatitis

Abstract

Tauhe expression of the critical initiator cytokine TNF-alpha was strongly upregulated in vivo in acute necrotic pancreatitis (AP) in rodents and in vitro in TNF-alpha activated acinar AR42J cells. Upregulation of tnf-alpha, inos, icam-1 and il-6 occurred both in TNF-alpha receptor 1 and 2 knock-out mice, but not in TNF-alpha knock-out mice, in cerulein-induced acute pancreatitis. Chromatin immunoprecipitation analysis showed that transcriptional factors (ELK-1, SP1, NF-kappaB and EGR-1) and chromatin modification complexes (HDAC1, HDAC2, GCN5, PCAF and CBP) were recruited and/or released from the promoter in a strictly ordered mechanism. Activation of tnf-alpha gene was also accompanied by an ordered increased level of histone H3K9, H3K14 and H3K18-acetylation and H3K4 methylation, as well as H4K5 acetylation. A better knowledge of the molecular mechanisms that control tnf-alpha gene regulation will provide deeper understanding of the initiation and development of the inflammatory processes occurring in acute pancreatitis triggered by TNF-alpha cytokine.

Fig. 1

Expression of tumor necrosis factor alpha (tnf-α), inducible nitric oxide synthase (inos), intercellular adhesion molecule 1 (icam-1) and interleukin 6 (il-6) in pancreas from a wild-type mice and TNF-α receptor 1 or 2 knock-out mice, and b TNF-α knock-out mice with cerulein-induced AP. Steady-state mRNA levels of tnf-α, inos, icam-1 and il-6 were measured by quantitative RT-PCR in pancreas from mice treated with saline (control) or cerulein as indicated in Methods. The error bars correspond with the standard deviation of 4–5 independent RT-PCR measurements. The statistical significance is indicated as follows: **P < 0.01 or *P < 0.05 vs. control or time 0

Fig. 2

The tnf-α gene expression in taurocholate-induced AP in rats. a RNApol ChIP analysis. α- and β-Actin genes were used as negative and positive control, respectively, of the RNApol ChIP assay. b Semiquantitative RT-PCR and c quantitative RT-PCR analysis of the tnf-α mRNA level. The rRNA 18S was used as an internal control, and α- and β-actin genes were used as negative and positive control, respectively, of the RT-PCR analysis. In the histogram, the quantitative RT-PCR values obtained for tnf-α or β-actin analysis were normalized against those obtained for rRNA 18S analysis, giving an arbitrary value of 1 to the non-treated sample. The error bars correspond with the standard deviation of at least three independent RT-PCR measurements. The statistical significance is indicated as follows: **P < 0.01 or *P < 0.05 vs. control or time 0. d Immunohistochemical detection of TNF-α production by acinar cells in rat pancreas 3 h after AP induction. Immunostaining negative control with tissue treated in absence of primary TNF-α antibody is also shown

Fig. 3

ChIP assay of tnf-α promoter occupation in taurocholate-induced AP in rats. a Putative binding sites for transcriptional factors in the tnf-α promoter analyzed by MatInspector Software. b The immunoprecipitated samples with indicated antibodies were analyzed by PCR using primers of the tnf-α promoter region. c ImageJ analysis of PCR signals. α-Actin gene has also been included as a negative control of the ChIP experiment

Fig. 4

The tnf-α gene expression in TNF-α-activated AR42J cells. a RNApol ChIP analysis. b Semiquantitative RT-PCR and c quantitative RT-PCR analysis of the tnf-α mRNA level. rRNA 18S was used as an internal control and β-actin as negative control of the RT-PCR analysis and α-actin as negative control of ChIP assay. The statistical significance is indicated as follows: **P < 0.01 or *P < 0.05 vs. control or time 0

Fig. 5

a ChIP assay of tnf-α promoter occupation in TNF-α-activated AR42J cells. b ImageJ analysis of PCR signals. The α-actin gene has been included as negative control of the ChIP experiment

Fig. 6

ChIP assay of chromatin-modifying complex occupation (a, b) and site-specific histone modifications (c–e) in tnf-α promoter in TNF-α-activated AR42J cells. The α-actin gene has been included as negative control for the ChIP experiment