Differential targeting of the E-Cadherin/β-Catenin complex by gram-positive probiotic lactobacilli improves epithelial barrier function - PubMed (original) (raw)

Differential targeting of the E-Cadherin/β-Catenin complex by gram-positive probiotic lactobacilli improves epithelial barrier function

Stephanie Hummel et al. Appl Environ Microbiol. 2012 Feb.

Abstract

The intestinal ecosystem is balanced by dynamic interactions between resident and incoming microbes, the gastrointestinal barrier, and the mucosal immune system. However, in the context of inflammatory bowel diseases (IBD), where the integrity of the gastrointestinal barrier is compromised, resident microbes contribute to the development and perpetuation of inflammation and disease. Probiotic bacteria have been shown to exert beneficial effects, e.g., enhancing epithelial barrier integrity. However, the mechanisms underlying these beneficial effects are only poorly understood. Here, we comparatively investigated the effects of four probiotic lactobacilli, namely, Lactobacillus acidophilus, L. fermentum, L. gasseri, and L. rhamnosus, in a T84 cell epithelial barrier model. Results of DNA microarray experiments indicating that lactobacilli modulate the regulation of genes encoding in particular adherence junction proteins such as E-cadherin and β-catenin were confirmed by quantitative reverse transcription-PCR (qRT-PCR). Furthermore, we show that epithelial barrier function is modulated by Gram-positive probiotic lactobacilli via their effect on adherence junction protein expression and complex formation. In addition, incubation with lactobacilli differentially influences the phosphorylation of adherence junction proteins and the abundance of protein kinase C (PKC) isoforms such as PKCδ that thereby positively modulates epithelial barrier function. Further insight into the underlying molecular mechanisms triggered by these probiotics might also foster the development of novel strategies for the treatment of gastrointestinal diseases (e.g., IBD).

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Figures

Fig 1

Fig 1

Monitoring changes of the transepithelial electrical resistance (TER) of T84 cell monolayers following incubation with lactobacilli and the enteropathogenic E. coli (EPEC) strain E2348/69. T84 cell monolayers were incubated with L. fermentum (L. f.) (A), L. gasseri (L. g.) (A), L. acidophilus (L. a.) (B), L. rhamnosus (L. r.) (B), and the enteropathogenic E. coli strain E2348/69 (EPEC) (C). T84 cells were grown on Transwell filter units, and the TER of untreated T84 cell monolayers (T84) and of T84 cell monolayers incubated with the different bacteria was measured. TER measurements were performed online using the cellZscope set-up (NanoAnalytics, Münster, Germany). The vertical line indicates the time when bacteria were added. TER values of untreated T84 cell monolayers were set to 100%.

Fig 2

Fig 2

Impact of incubation of T84 cells with different lactobacilli on ZO-2 (TJ-associated protein) and E-cadherin (AJ-associated protein). T84 cells were incubated with L. gasseri, L. acidophilus, L. fermentum, and L. rhamnosus for 30, 60, 120, and 180 min. ZO-2 and E-cadherin mRNA expression was quantified using qRT-PCR (*, P ≤ 0.05). Bars represent standard deviations (SD).

Fig 3

Fig 3

Western blot analysis of E-cadherin and ZO-2 expression following incubation of T84 cells with lactobacilli. T84 cells were incubated with L. acidophilus, L. fermentum, L. gasseri, and L. rhamnosus for 30, 60, 120, 180, and 240 min. Protein expression in T84 cells that were incubated with lactobacilli relative to that in untreated T84 cells is shown. Representative blots of experiments conducted at least three times are shown. Densitometry measurements, including SD, are given below the gels. The values for untreated T84 cells were set to 1.

Fig 4

Fig 4

Distribution and phosphorylation of PKCδ in T84 cells after incubation with lactobacilli. Following incubation of T84 cells with L. fermentum, L. rhamnosus, L. gasseri, and L. acidophilus for 2.5 h, cells were fractionated in cytosolic (CF) and membrane (MF) fractions. Following Western blotting, probing with anti-P-Ser/Thr antibodies, development, and documentation (A), the membranes were stripped and probed additionally with anti-PKCδ specific antibodies (B). T84 fractions were probed with anti-PKCδ antibodies (C). (D) α-Tubulin loading control. Following incubation with the different lactobacillus species, the abundance of P-PKCδ in the membrane fraction of T84 cells (•) is reduced (○). Relevant positions are indicated by an arrowhead. Representative blots from experiments performed at least three times are shown.

Fig 5

Fig 5

Phosphorylation status of PKCζ in T84 cells after incubation with L. fermentum, L. rhamnosus, L. gasseri, and L. acidophilus for 2.5 h. Protein expression in T84 cells is depicted in comparison to that in T84 cells that were not incubated with lactobacilli. Probing and development was performed as described in the legend to Fig. 4. Representative blots of experiments repeated at least three times are shown. CF, cytosolic fraction; MF, membrane fraction. Relative positions are indicated by an arrowhead.

Fig 6

Fig 6

Phosphorylation of β-catenin after incubation of T84 cells with L. fermentum, L. rhamnosus, L. gasseri, and L. acidophilus for 2.5 h. Cellular fractionation in cytoplasmic (CF) and membrane (MF) fractions indicated a differentially enhanced expression of Ser/Thr-phosphorylated β-catenin in the membrane fractions of T84 cells induced by incubation with different lactobacilli.

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