Intestinal Alkaline Phosphatase Regulates Tight Junction Protein Levels

Abstract

Background: Intestinal alkaline phosphatase (IAP) plays a pivotal role in maintaining gut health and well-being. Oral supplementation with IAP in mice improves gut barrier function and prevents luminal proinflammatory factors from gaining access to the circulation. In this study, we sought to explore the relationship between IAP and tight junction protein (TJP) expression and function.

Study design: The effect of IAP deletion on TJP levels was studied in mouse embryonic fibroblasts (MEFs) generated from IAP-knockout and wild type mice. Regulation of TJPs by IAP was assayed in the human colon cancer Caco-2 and T84 cells by overexpressing the human IAP gene. Tight junction protein levels and localization were measured by using RT q-PCR and antibodies targeting the specific TJPs. Finally, the effect of IAP on inflammation-induced intestinal permeability was measured by in vitro trans-well epithelial electrical resistance (TEER).

Results: Intestinal alkaline phosphatase gene deletion in MEFs resulted in significantly lower levels of ZO-1, ZO-2, and Occludin compared with levels in wild-type control cells; IAP overexpression in Caco-2 and T84 cells resulted in approximate 2-fold increases in the mRNA levels of ZO-1 and ZO-2. The IAP treatment ameliorated lipopolysaccharide-induced increased permeability in the Caco-2 trans-well system. Furthermore, IAP treatment preserved the localization of the ZO-1 and Occludin proteins during inflammation and was also associated with improved epithelial barrier function.

Conclusions: Intestinal alkaline phosphatase is a major regulator of gut mucosal permeability and appears to work at least partly through improving TJP levels and localization. These data provide a strong foundation to develop IAP as a novel therapy to maintain gut barrier function.

Figure 1

Mouse embryonic fibroblasts (MEFs) from intestinal alkaline phosphatase (IAP)-knockout mice have less tight junction protein levels. The MEFs from wild-type (WT) and IAP-knockout (KO) mice were seeded at a density of 3 × 10⁵ cells per well in 6-well plate (n = 3). At 80% confluence, RNA was isolated using Trizole, and quantitative real-time polymerase chain reaction (qRT-PCR) was performed to determine the levels of tight junction protein expression. Tight junction protein levels in MEFs from IAP-KO mice were compared with those of WT. Values are expressed as mean ± SEM. Statistical significance between the 2 groups was tested using the 2-tailed Student’s t-test. *p < 0.05.

Figure 2

Intestinal alkaline phosphatase (IAP) abundance increases tight junction protein (TJP) levels in the human colon cancer Caco-2 and T84 cell lines. Stably transfected cells with the human IAP gene or with the empty vector were used to study the effect of IAP overexpression on TJP levels. Cells were seeded in 6-well plates in triplicate. After formation of monolayer, changes in mRNA levels were assayed using quantitative real-time polymerase chain reaction (qRT-PCR). Results from IAP overexpressing cells were compared with those from cells transfected with the empty vector: (A) Human IAP relative expression and junctional protein mRNA expression in (B) Caco-2 and (C) T84 cell lines. Values are expressed as mean ± SEM. Statistical significance between the 2 groups was tested using the 2-tailed Student’s t-test. **p < 0.01, ***p < 0.001.

Figure 3

Exogenous intestinal alkaline phosphatase (IAP) treatment prevents lipopolysaccharide (LPS)-induced decrease in tight junction protein levels. Caco-2 cells were seeded at a density of 3 × 10⁵ cells per well in 6-well plates. After forming the monolayer, around 48 hours, LPS (100 ng/mL) + IAP 500 U or vehicle (VEH) or equal amount of PBS as control were added to the wells in triplicates. Twenty-four hours later, RNA and proteins were isolated for tight junction protein quantification. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to determine the levels of tight junction protein expression. (A) Junctional protein mRNA expression in Caco-2 cells treated with LPS ± IAP. In the same experiment, ZO-1 protein levels were measured by Western blot using antibody against human ZO-1 and normalized the levels of beta-actin in the same samples. (B) Western blot determinations of ZO-1 levels in Caco-2 cells treated with LPS ± IAP. Values are expressed as mean ± SEM. Statistical significance between the groups was tested using 1-way analysis of variance with Tukey’s multiple comparison post-tests. **p < 0.01, ***p < 0.001.

Figure 4

Intestinal alkaline phosphatase (IAP) treatment prevents lipopolysaccharide (LPS)-induced barrier dysfunction in Caco-2 trans-well system. Caco-2 cells were seeded onto the filter supports in the trans-well at a concentration of 3 × 10⁵ cells/insert and were incubated in cell culture incubator (37°C, 5% CO₂). At a resistance reading of 200 Ω.cm² by transepithelial electrical resistance (TEER) in the Caco-2 trans-wells, LPS 100 ng/mL ± IAP 500 U/mL or vehicle (VEH) were added to both compartments of the trans-well with fresh growth media in triplicate daily. Equal amounts of phosphate buffered saline (PBS) were added as a control. The TEER readings were documented daily for 5 days. Values are expressed as mean ± SEM. Statistical significance between the groups was tested using 1-way analysis of variance with Tukey’s multiple comparison post-tests. **p < 0.01, ***p < 0.001.

Figure 5

Intestinal alkaline phosphatase (IAP) treatment preserve tight junctions’ integrity. Caco-2 cells were seeded onto the filter supports in the trans-well at a concentration of 3 × 10⁵ cells/insert and were incubated in cell culture incubator (37°C, 5% CO₂). After forming the monolayer, lipopolysaccharide (LPS) 100 ng/mL ± IAP 500 U/mL or vehicle (VEH) were added to both compartments of the trans-well with fresh growth media in triplicate daily. Equal amounts of phosphate buffered saline (PBS) were added as a control. After 5 days, inserts were removed from wells, and cells were fixed with 4% paraformaldehyde. Localization of ZO-1 and Occludin proteins in the same cells were detected by immunofluorescence using primary antibody against human ZO-1 and Occludin. Secondary antibodies with different fluorescence wavelength were used for the indirect detection of different antigens. (Top) ZO-1 distribution and (bottom) Occludin distribution.

Figure 6

Intestinal alkaline phosphatase (IAP) treatment prevents lipopolysaccharide (LPS)-induced cytokine production. Caco-2 cells were seeded onto the filter supports in the trans-well at a concentration of 3 × 10⁵ cells/insert. After forming the monolayer, LPS 100 ng/mL ± IAP 500 U/mL or vehicle (VEH) were added to both compartments of the trans-well with fresh growth media in triplicate daily. After 5 days, RNA was isolated using Trizole and quantitative real-time polymerase chain reaction (qRT-PCR) was performed to determine the levels of inflammatory cytokines: (A) tumor necrosis factor (TNF)-α, (B) interleukin (IL)-1β, and (C) IL-8 expression levels. Values are expressed as mean ± SEM. Statistical significance between the groups was tested using 1-way analysis of variance with Tukey’s multiple comparison post-tests. *p < 0.05, **p < 0.01.