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Tight junction disruption by cadmium in an in vitro human airway tissue model.

Cao X, Lin H, Muskhelishvili L, Latendresse J, Richter P, Heflich RH - Respir. Res. (2015)

Bottom Line: Noncytotoxic doses of CdCl2 resulted in the collapse of barrier function, as demonstrated by TEER measurements and Zonula occludens-1 (ZO-1) and occludin staining.Inhibition of kinase signaling using inhibitors specific for cellular Src or PKC preserved the integrity of TJs, possibly by preventing occludin tyrosine hyperphosphorylation, rather than reversing the down-regulation of the junction-interacting proteins.Our findings indicate that acute doses of Cd likely disrupt TJ integrity in human ALI airway cultures both through occludin hyperphosphorylation via kinase activation and by direct disruption of the junction-interacting complex.

View Article: PubMed Central - PubMed

ABSTRACT

Background: The cadmium (Cd) present in air pollutants and cigarette smoke has the potential of causing multiple adverse health outcomes involving damage to pulmonary and cardiovascular tissue. Injury to pulmonary epithelium may include alterations in tight junction (TJ) integrity, resulting in impaired epithelial barrier function and enhanced penetration of chemicals and biomolecules. Herein, we investigated mechanisms involved in the disruption of TJ integrity by Cd exposure using an in vitro human air-liquid-interface (ALI) airway tissue model derived from normal primary human bronchial epithelial cells.

Methods: ALI cultures were exposed to noncytotoxic doses of CdCl2 basolaterally and TJ integrity was measured by Trans-Epithelial Electrical Resistance (TEER) and immunofluorescence staining with TJ markers. PCR array analysis was used to identify genes involved with TJ collapse. To explore the involvement of kinase signaling pathways, cultures were treated with CdCl2 in the presence of kinase inhibitors specific for cellular Src or Protein Kinase C (PKC).

Results: Noncytotoxic doses of CdCl2 resulted in the collapse of barrier function, as demonstrated by TEER measurements and Zonula occludens-1 (ZO-1) and occludin staining. CdCl2 exposure altered the expression of several groups of genes encoding proteins involved in TJ homeostasis. In particular, down-regulation of select junction-interacting proteins suggested that a possible mechanism for Cd toxicity involves disruption of the peripheral junctional complexes implicated in connecting membrane-bound TJ components to the actin cytoskeleton. Inhibition of kinase signaling using inhibitors specific for cellular Src or PKC preserved the integrity of TJs, possibly by preventing occludin tyrosine hyperphosphorylation, rather than reversing the down-regulation of the junction-interacting proteins.

Conclusions: Our findings indicate that acute doses of Cd likely disrupt TJ integrity in human ALI airway cultures both through occludin hyperphosphorylation via kinase activation and by direct disruption of the junction-interacting complex.

No MeSH data available.


Related in: MedlinePlus

Cingulin, TJAP1, and VAP-33 protein expression changes in response to CdCl2treatment. Cultures were treated from the basolateral side with various concentrations of CdCl2 for 24 h, and whole cell lysate was collected and separated by electrophoresis. The expression of cingulin, TJAP1, and VAP-33 was detected by immunoblotting using specific antibodies. (A). Representative Western blots. (B). Density of the bands were quantified and statistically analyzed (N = 3). *Indicates p < 0.05 compared to the vehicle-treated control.
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Fig6: Cingulin, TJAP1, and VAP-33 protein expression changes in response to CdCl2treatment. Cultures were treated from the basolateral side with various concentrations of CdCl2 for 24 h, and whole cell lysate was collected and separated by electrophoresis. The expression of cingulin, TJAP1, and VAP-33 was detected by immunoblotting using specific antibodies. (A). Representative Western blots. (B). Density of the bands were quantified and statistically analyzed (N = 3). *Indicates p < 0.05 compared to the vehicle-treated control.

Mentions: Since genes involved in the junctional-protein complex were among the top gene groups modulated by 100 μM CdCl2, three differentially expressed junctional-interacting genes, e.g., cingulin, tight junction interaction protein 1 (TJAP1), and VAP-33, were selected and changes in their protein expression were further examined by immunoblotting. Cingulin and TJAP1 exhibited clear dose-dependent decreases in their protein expression with increasing concentrations of CdCl2; the expression of VAP-33 also was decreased but its decrease was less dose-responsive (Figure 6A). Quantitative assessment, however, indicated that treatment with 100 μM CdCl2 decreased the expression of all three junctional-interacting proteins by approximately 50%, decreases that were statistically significant (Figure 6B).Figure 6


Tight junction disruption by cadmium in an in vitro human airway tissue model.

Cao X, Lin H, Muskhelishvili L, Latendresse J, Richter P, Heflich RH - Respir. Res. (2015)

Cingulin, TJAP1, and VAP-33 protein expression changes in response to CdCl2treatment. Cultures were treated from the basolateral side with various concentrations of CdCl2 for 24 h, and whole cell lysate was collected and separated by electrophoresis. The expression of cingulin, TJAP1, and VAP-33 was detected by immunoblotting using specific antibodies. (A). Representative Western blots. (B). Density of the bands were quantified and statistically analyzed (N = 3). *Indicates p < 0.05 compared to the vehicle-treated control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4352288&req=5

Fig6: Cingulin, TJAP1, and VAP-33 protein expression changes in response to CdCl2treatment. Cultures were treated from the basolateral side with various concentrations of CdCl2 for 24 h, and whole cell lysate was collected and separated by electrophoresis. The expression of cingulin, TJAP1, and VAP-33 was detected by immunoblotting using specific antibodies. (A). Representative Western blots. (B). Density of the bands were quantified and statistically analyzed (N = 3). *Indicates p < 0.05 compared to the vehicle-treated control.
Mentions: Since genes involved in the junctional-protein complex were among the top gene groups modulated by 100 μM CdCl2, three differentially expressed junctional-interacting genes, e.g., cingulin, tight junction interaction protein 1 (TJAP1), and VAP-33, were selected and changes in their protein expression were further examined by immunoblotting. Cingulin and TJAP1 exhibited clear dose-dependent decreases in their protein expression with increasing concentrations of CdCl2; the expression of VAP-33 also was decreased but its decrease was less dose-responsive (Figure 6A). Quantitative assessment, however, indicated that treatment with 100 μM CdCl2 decreased the expression of all three junctional-interacting proteins by approximately 50%, decreases that were statistically significant (Figure 6B).Figure 6

Bottom Line: Noncytotoxic doses of CdCl2 resulted in the collapse of barrier function, as demonstrated by TEER measurements and Zonula occludens-1 (ZO-1) and occludin staining.Inhibition of kinase signaling using inhibitors specific for cellular Src or PKC preserved the integrity of TJs, possibly by preventing occludin tyrosine hyperphosphorylation, rather than reversing the down-regulation of the junction-interacting proteins.Our findings indicate that acute doses of Cd likely disrupt TJ integrity in human ALI airway cultures both through occludin hyperphosphorylation via kinase activation and by direct disruption of the junction-interacting complex.

View Article: PubMed Central - PubMed

ABSTRACT

Background: The cadmium (Cd) present in air pollutants and cigarette smoke has the potential of causing multiple adverse health outcomes involving damage to pulmonary and cardiovascular tissue. Injury to pulmonary epithelium may include alterations in tight junction (TJ) integrity, resulting in impaired epithelial barrier function and enhanced penetration of chemicals and biomolecules. Herein, we investigated mechanisms involved in the disruption of TJ integrity by Cd exposure using an in vitro human air-liquid-interface (ALI) airway tissue model derived from normal primary human bronchial epithelial cells.

Methods: ALI cultures were exposed to noncytotoxic doses of CdCl2 basolaterally and TJ integrity was measured by Trans-Epithelial Electrical Resistance (TEER) and immunofluorescence staining with TJ markers. PCR array analysis was used to identify genes involved with TJ collapse. To explore the involvement of kinase signaling pathways, cultures were treated with CdCl2 in the presence of kinase inhibitors specific for cellular Src or Protein Kinase C (PKC).

Results: Noncytotoxic doses of CdCl2 resulted in the collapse of barrier function, as demonstrated by TEER measurements and Zonula occludens-1 (ZO-1) and occludin staining. CdCl2 exposure altered the expression of several groups of genes encoding proteins involved in TJ homeostasis. In particular, down-regulation of select junction-interacting proteins suggested that a possible mechanism for Cd toxicity involves disruption of the peripheral junctional complexes implicated in connecting membrane-bound TJ components to the actin cytoskeleton. Inhibition of kinase signaling using inhibitors specific for cellular Src or PKC preserved the integrity of TJs, possibly by preventing occludin tyrosine hyperphosphorylation, rather than reversing the down-regulation of the junction-interacting proteins.

Conclusions: Our findings indicate that acute doses of Cd likely disrupt TJ integrity in human ALI airway cultures both through occludin hyperphosphorylation via kinase activation and by direct disruption of the junction-interacting complex.

No MeSH data available.


Related in: MedlinePlus