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Toll-like receptor 2 regulates the barrier function of human bronchial epithelial monolayers through atypical protein kinase C zeta, and an increase in expression of claudin-1.

Ragupathy S, Esmaeili F, Paschoud S, Sublet E, Citi S, Borchard G - Tissue Barriers (2014)

Bottom Line: This was confirmed by a decrease in paracellular flux of fluorescein sodium.This TLR2 induced increase in TEER was significantly reduced by pretreatment with polyclonal anti-human TLR2-neutralizing antibody.TLR2 stimulation in Calu-3 cell monolayers resulted in an increased expression of the tight junction proteins claudin-1 and ZO-1, and a decreased expression of occludin, at both the mRNA and protein levels.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmaceutical Sciences; University of Geneva; University of Lausanne; Geneva, Switzerland.

ABSTRACT
We investigated the role of Toll-like receptor (TLR) 2 in maintaining the integrity of the airway epithelial barrier using the human bronchial epithelial cell line Calu-3. Activation of TLR2 by its ligands, Pam3CysSK4 and Peptidoglycan showed a concentration dependent increase in epithelial barrier function, as measured by transepithelial electrical resistance (TEER). This was confirmed by a decrease in paracellular flux of fluorescein sodium. This TLR2 induced increase in TEER was significantly reduced by pretreatment with polyclonal anti-human TLR2-neutralizing antibody. TLR2 stimulation in Calu-3 cell monolayers resulted in an increased expression of the tight junction proteins claudin-1 and ZO-1, and a decreased expression of occludin, at both the mRNA and protein levels. A pseudosubstrate inhibitor to PKCζ significantly prevented the TLR2 mediated increase in barrier function. It also prevented the increase in claudin-1 in a concentration dependent manner up to 1 µM. TLR2 stimulation led to an increase in phosphorylation of atypical PKC ζ, which was prevented by the pseudosubstrate inhibitor in a concentration dependent manner. Taken together, our observations support a model whereby increased tight junction barrier function induced by activation of TLR2 occurs through increased expression of claudin-1, and through modulation of PKC ζ activity.

No MeSH data available.


Related in: MedlinePlus

Figure 4. (A) Pretreatment with 25 µM PKC ζ pseudosubstrate inhibitor (PSI) significantly prevented the increase in TEER induced by PGN. All TEER values are expressed as percentage relative to control (the baseline value was > 520 Ω*cm2). Values represent mean ± SD *P < 0.05, **P < 0.01, ***P < 0.001. The data were analyzed using one-way analysis of variance (ANOVA). All the experiments were performed atleast 3 times during different days with number of replicates (n = 4). Western blotting (B) for claudin-1, occludin and ZO-1. Upregulation of claudin-1 and ZO-1 was observed in Calu-3 cells after treatment with P3C and PGN. Occludin was decreased. The images are representative of 3 independent experiments. β–actin was used as a lane loading control. The number of replicates is (n = 2). (C) the corresponding expression levels of claudin-1, occluding and ZO-1 are shown as bar graphs. (D) western blotting for claudin-1. The PSI inhibited the claudin-1 expression induced by PGN treatment in a concentration dependent manner (25 µM, 10 µM and 1 µM). E, the corresponding expression levels of claudin-1 are shown as bar graphs. (F) western blotting for phospho-PKC ζ in Calu-3 cell monolayers treated with PGN. Stimulation by PGN increased phosphorylation of PKC ζ in the treated cells which was inhibited by pretreatment with PSI 25 µM and 10 μM. Expression of phospho-PKC ζ and total PKC ζ was examined using western-blot analyses of total cell lysates. The images are representative of 3 independent experiments. The number of replicates is (n = 2). (G) the corresponding expression levels of phospho-PKC ζ are shown as relative to total PKC ζ expression.
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Figure 4: Figure 4. (A) Pretreatment with 25 µM PKC ζ pseudosubstrate inhibitor (PSI) significantly prevented the increase in TEER induced by PGN. All TEER values are expressed as percentage relative to control (the baseline value was > 520 Ω*cm2). Values represent mean ± SD *P < 0.05, **P < 0.01, ***P < 0.001. The data were analyzed using one-way analysis of variance (ANOVA). All the experiments were performed atleast 3 times during different days with number of replicates (n = 4). Western blotting (B) for claudin-1, occludin and ZO-1. Upregulation of claudin-1 and ZO-1 was observed in Calu-3 cells after treatment with P3C and PGN. Occludin was decreased. The images are representative of 3 independent experiments. β–actin was used as a lane loading control. The number of replicates is (n = 2). (C) the corresponding expression levels of claudin-1, occluding and ZO-1 are shown as bar graphs. (D) western blotting for claudin-1. The PSI inhibited the claudin-1 expression induced by PGN treatment in a concentration dependent manner (25 µM, 10 µM and 1 µM). E, the corresponding expression levels of claudin-1 are shown as bar graphs. (F) western blotting for phospho-PKC ζ in Calu-3 cell monolayers treated with PGN. Stimulation by PGN increased phosphorylation of PKC ζ in the treated cells which was inhibited by pretreatment with PSI 25 µM and 10 μM. Expression of phospho-PKC ζ and total PKC ζ was examined using western-blot analyses of total cell lysates. The images are representative of 3 independent experiments. The number of replicates is (n = 2). (G) the corresponding expression levels of phospho-PKC ζ are shown as relative to total PKC ζ expression.

Mentions: Next, we evaluated the effect of TLR2 stimulation on the expression and localization of TJ proteins. In Calu-3 cells, RT-PCR analysis revealed an upregulation of claudin-1 and ZO-1 mRNA by about 200-fold and 30-fold increase, respectively, when compared with the control (Fig. 3A). In contrast, the expression of claudin-2 and occludin was decreased about 50-fold and 70-fold, respectively (Fig. 3A). Immunofluoresence analysis showed that treatment with P3C leads to an increase in the junctional labeling of claudin-1 and ZO-1, a decrease in occludin labeling and no effect on cingulin labeling (Fig. 3B). Western blotting analysis for claudin-1, ZO-1 and occludin showed an increase in the levels of claudin-1 and ZO-1 while a decrease in occludin compared with control. (Fig. 4B and C).


Toll-like receptor 2 regulates the barrier function of human bronchial epithelial monolayers through atypical protein kinase C zeta, and an increase in expression of claudin-1.

Ragupathy S, Esmaeili F, Paschoud S, Sublet E, Citi S, Borchard G - Tissue Barriers (2014)

Figure 4. (A) Pretreatment with 25 µM PKC ζ pseudosubstrate inhibitor (PSI) significantly prevented the increase in TEER induced by PGN. All TEER values are expressed as percentage relative to control (the baseline value was > 520 Ω*cm2). Values represent mean ± SD *P < 0.05, **P < 0.01, ***P < 0.001. The data were analyzed using one-way analysis of variance (ANOVA). All the experiments were performed atleast 3 times during different days with number of replicates (n = 4). Western blotting (B) for claudin-1, occludin and ZO-1. Upregulation of claudin-1 and ZO-1 was observed in Calu-3 cells after treatment with P3C and PGN. Occludin was decreased. The images are representative of 3 independent experiments. β–actin was used as a lane loading control. The number of replicates is (n = 2). (C) the corresponding expression levels of claudin-1, occluding and ZO-1 are shown as bar graphs. (D) western blotting for claudin-1. The PSI inhibited the claudin-1 expression induced by PGN treatment in a concentration dependent manner (25 µM, 10 µM and 1 µM). E, the corresponding expression levels of claudin-1 are shown as bar graphs. (F) western blotting for phospho-PKC ζ in Calu-3 cell monolayers treated with PGN. Stimulation by PGN increased phosphorylation of PKC ζ in the treated cells which was inhibited by pretreatment with PSI 25 µM and 10 μM. Expression of phospho-PKC ζ and total PKC ζ was examined using western-blot analyses of total cell lysates. The images are representative of 3 independent experiments. The number of replicates is (n = 2). (G) the corresponding expression levels of phospho-PKC ζ are shown as relative to total PKC ζ expression.
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Related In: Results  -  Collection

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Figure 4: Figure 4. (A) Pretreatment with 25 µM PKC ζ pseudosubstrate inhibitor (PSI) significantly prevented the increase in TEER induced by PGN. All TEER values are expressed as percentage relative to control (the baseline value was > 520 Ω*cm2). Values represent mean ± SD *P < 0.05, **P < 0.01, ***P < 0.001. The data were analyzed using one-way analysis of variance (ANOVA). All the experiments were performed atleast 3 times during different days with number of replicates (n = 4). Western blotting (B) for claudin-1, occludin and ZO-1. Upregulation of claudin-1 and ZO-1 was observed in Calu-3 cells after treatment with P3C and PGN. Occludin was decreased. The images are representative of 3 independent experiments. β–actin was used as a lane loading control. The number of replicates is (n = 2). (C) the corresponding expression levels of claudin-1, occluding and ZO-1 are shown as bar graphs. (D) western blotting for claudin-1. The PSI inhibited the claudin-1 expression induced by PGN treatment in a concentration dependent manner (25 µM, 10 µM and 1 µM). E, the corresponding expression levels of claudin-1 are shown as bar graphs. (F) western blotting for phospho-PKC ζ in Calu-3 cell monolayers treated with PGN. Stimulation by PGN increased phosphorylation of PKC ζ in the treated cells which was inhibited by pretreatment with PSI 25 µM and 10 μM. Expression of phospho-PKC ζ and total PKC ζ was examined using western-blot analyses of total cell lysates. The images are representative of 3 independent experiments. The number of replicates is (n = 2). (G) the corresponding expression levels of phospho-PKC ζ are shown as relative to total PKC ζ expression.
Mentions: Next, we evaluated the effect of TLR2 stimulation on the expression and localization of TJ proteins. In Calu-3 cells, RT-PCR analysis revealed an upregulation of claudin-1 and ZO-1 mRNA by about 200-fold and 30-fold increase, respectively, when compared with the control (Fig. 3A). In contrast, the expression of claudin-2 and occludin was decreased about 50-fold and 70-fold, respectively (Fig. 3A). Immunofluoresence analysis showed that treatment with P3C leads to an increase in the junctional labeling of claudin-1 and ZO-1, a decrease in occludin labeling and no effect on cingulin labeling (Fig. 3B). Western blotting analysis for claudin-1, ZO-1 and occludin showed an increase in the levels of claudin-1 and ZO-1 while a decrease in occludin compared with control. (Fig. 4B and C).

Bottom Line: This was confirmed by a decrease in paracellular flux of fluorescein sodium.This TLR2 induced increase in TEER was significantly reduced by pretreatment with polyclonal anti-human TLR2-neutralizing antibody.TLR2 stimulation in Calu-3 cell monolayers resulted in an increased expression of the tight junction proteins claudin-1 and ZO-1, and a decreased expression of occludin, at both the mRNA and protein levels.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmaceutical Sciences; University of Geneva; University of Lausanne; Geneva, Switzerland.

ABSTRACT
We investigated the role of Toll-like receptor (TLR) 2 in maintaining the integrity of the airway epithelial barrier using the human bronchial epithelial cell line Calu-3. Activation of TLR2 by its ligands, Pam3CysSK4 and Peptidoglycan showed a concentration dependent increase in epithelial barrier function, as measured by transepithelial electrical resistance (TEER). This was confirmed by a decrease in paracellular flux of fluorescein sodium. This TLR2 induced increase in TEER was significantly reduced by pretreatment with polyclonal anti-human TLR2-neutralizing antibody. TLR2 stimulation in Calu-3 cell monolayers resulted in an increased expression of the tight junction proteins claudin-1 and ZO-1, and a decreased expression of occludin, at both the mRNA and protein levels. A pseudosubstrate inhibitor to PKCζ significantly prevented the TLR2 mediated increase in barrier function. It also prevented the increase in claudin-1 in a concentration dependent manner up to 1 µM. TLR2 stimulation led to an increase in phosphorylation of atypical PKC ζ, which was prevented by the pseudosubstrate inhibitor in a concentration dependent manner. Taken together, our observations support a model whereby increased tight junction barrier function induced by activation of TLR2 occurs through increased expression of claudin-1, and through modulation of PKC ζ activity.

No MeSH data available.


Related in: MedlinePlus