The Pro-inflammatory Cytokines, IL-1βand TNF-α, Inhibit Intestinal Alkaline Phosphatase Gene Expression

Abstract

High levels of the pro-inflammatory cytokines, interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), are present in the gut mucosa of patients suffering form various diseases, most notably inflammatory bowel diseases (IBD). Since the inflammatory milieu can cause important alterations in epithelial cell function, we examined the cytokine effects on the expression of the enterocyte differentiation marker, intestinal alkaline phosphatase (IAP), a protein that detoxifies bacterial lipopolysaccharides (LPS) and limits fat absorption. Sodium butyrate (NaBu), a short-chain fatty acid and histone deacetylase (HDAC) inhibitor, was used to induce IAP expression in HT-29 cells and the cells were also treated +/- the cytokines. Northern blots confirmed IAP induction by NaBu, however, pretreatment (6 h) with either cytokine showed a dose-dependent inhibition of IAP expression. IAP Western analyses and alkaline phosphatase enzyme assays corroborated the Northern data and confirmed that the cytokines inhibit IAP induction. Transient transfections with a reporter plasmid carrying the human IAP promoter showed significant inhibition of NaBu-induced IAP gene activation by the cytokines (100 and 60% inhibition with IL-1beta and TNF-alpha, respectively). Western analyses showed that NaBu induced H4 and H3 histone acetylation, and pretreatment with IL-1beta or TNF-alpha did not change this global acetylation pattern. In contrast, chromatin immunoprecipitation showed that local histone acetylation of the IAP promoter region was specifically inhibited by either cytokine. We conclude that IL-1beta and TNF-alpha inhibit NaBu-induced IAP gene expression, likely by blocking the histone acetylation within its promoter. Cytokine-mediated IAP gene silencing may have important implications for gut epithelial function in the setting of intestinal inflammatory conditions.

Intestinal alkaline phosphatase and sodium butyrate may be beneficial in attenuating LPS-induced intestinal inflammation

“…However, there are no reports regarding the ability of exogenous IAP to regulate endogenous IAP expression. A few studies have suggested that herbal extracts and sodium butyrate may play a role in regulating IAP expression (Malo et al, 2006;Levkut et al, 2011). The present investigation demonstrated that EO, NaBu and exogenous IAP all up-regulate intestinal levels of IAP, which can contribute to the maintenance of local intestinal immunity.…”

“…In the present study, the reduction of p65 and increased IAP expression in response to LPS+NaBu treatment suggest that, at least in part, the overexpression of IAP may down-regulate NF-κB signaling. The mechanism of action was reported be through histone hyperacetylation (Malo et al, 2006). Other studies examining the effects of NaBu on intestinal inflammation with regard to other pathways were summarized by Canani et al (2011).…”

“…Recently, in vitro studies confirmed that NaBu up-regulates the gene expression and activity of the brush border enzyme intestinal alkaline phosphatase (IAP) (Malo et al, 2006;Bol-Schoenmakers et al, 2010). IAP is an isoenzyme that hydrolyzes phosphate monoesters at alkaline pH (Sussman et al, 1989), and is able to detoxify bacterial components such as LPS (Koyama et al, 2002;Bates et al, 2007), flagellin, and CpG DNA (Chen et al, 2010) via dephosphorylation.…”

Intestinal alkaline phosphatase is a gut mucosal defense factor maintained by enteral nutrition

“…Both the fractionated cell components and lysate of all cells used pNPP as the substrate for the AP enzyme. As reported (32), to determine IAP activity, spectrophotometric quantitation of the hydrolyzed product of pNPP, p-nitrophenol, was used and calculated as nmol of pNPP hydrolyzed per minute per microgram of protein. To confirm that the AP activity was only from IAP, samples were also exposed to 10 mM phenylalanine, a known inhibitor of IAP, and 10 mM homoarginine, known to have no effect on IAP activity.…”

Clinical pharmacology of exogenously administered alkaline phosphatase

“…A growing body of evidence suggests that AP plays an important part in host defense in dephosphorylating extracellular ATP [8,9], a known pro-inflammatory molecule, into adenosine, a key molecule in tissue protection mechanisms [10], while extracellular ATP is also involved in the activity of phosphatases and phosphodiesterases [11]. This ecto-enzyme also plays an important role against inflammatory reactions due to lipopolysaccharide (LPS endotoxin) release during bacterial infection [12,13].…”