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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

Morphological characterization of ALI human airway cultures. ALI cultures were fixed in 10% formalin 5 weeks after the initial seeding onto the permeable membrane support. Tissue sections were stained with H&E (A), p63 (B), Ki67 (C), and PAS (D). Examples of positively stained cells are indicated by arrows. The arrows in (A), (B), (C), and (D) indicate ciliated cells, basal cells, actively proliferating cells, and goblet cells, respectively.
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Fig1: Morphological characterization of ALI human airway cultures. ALI cultures were fixed in 10% formalin 5 weeks after the initial seeding onto the permeable membrane support. Tissue sections were stained with H&E (A), p63 (B), Ki67 (C), and PAS (D). Examples of positively stained cells are indicated by arrows. The arrows in (A), (B), (C), and (D) indicate ciliated cells, basal cells, actively proliferating cells, and goblet cells, respectively.

Mentions: The morphology of differentiated ALI cultures was evaluated by histological methods 5 weeks after the initial seeding onto the permeable membrane support. H&E staining of the paraffin-embedded tissue sections indicated that the ALI cultures were fully differentiated into a pseudostratified mucociliary epithelium, with cells resembling goblet cells interspersed among ciliated cells along the apical side of the cultures (Figure 1A). Different types of epithelial cells were further distinguished by cell-specific markers. The cuboidal-shaped basal cells were identified by staining with the progenitor cell marker, p63 (Figure 1B). Cells expressing p63 were found exclusively along the basolateral side of the ALI culture. Cells undergoing active proliferation also were located only on the basolateral side, as demonstrated by staining with the cellular proliferation marker, Ki-67 (Figure 1C). PAS staining, which detects the glycoprotein and glycolipid components in mucin, was used to identify the mucus-secreting goblet cells (Figure 1D). Consistent with the H&E staining, PAS-positive goblet cells were distributed along the apical side of the culture. Residual secreted mucus also was detected on the apical side by PAS staining.Figure 1


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)

Morphological characterization of ALI human airway cultures. ALI cultures were fixed in 10% formalin 5 weeks after the initial seeding onto the permeable membrane support. Tissue sections were stained with H&E (A), p63 (B), Ki67 (C), and PAS (D). Examples of positively stained cells are indicated by arrows. The arrows in (A), (B), (C), and (D) indicate ciliated cells, basal cells, actively proliferating cells, and goblet cells, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Morphological characterization of ALI human airway cultures. ALI cultures were fixed in 10% formalin 5 weeks after the initial seeding onto the permeable membrane support. Tissue sections were stained with H&E (A), p63 (B), Ki67 (C), and PAS (D). Examples of positively stained cells are indicated by arrows. The arrows in (A), (B), (C), and (D) indicate ciliated cells, basal cells, actively proliferating cells, and goblet cells, respectively.
Mentions: The morphology of differentiated ALI cultures was evaluated by histological methods 5 weeks after the initial seeding onto the permeable membrane support. H&E staining of the paraffin-embedded tissue sections indicated that the ALI cultures were fully differentiated into a pseudostratified mucociliary epithelium, with cells resembling goblet cells interspersed among ciliated cells along the apical side of the cultures (Figure 1A). Different types of epithelial cells were further distinguished by cell-specific markers. The cuboidal-shaped basal cells were identified by staining with the progenitor cell marker, p63 (Figure 1B). Cells expressing p63 were found exclusively along the basolateral side of the ALI culture. Cells undergoing active proliferation also were located only on the basolateral side, as demonstrated by staining with the cellular proliferation marker, Ki-67 (Figure 1C). PAS staining, which detects the glycoprotein and glycolipid components in mucin, was used to identify the mucus-secreting goblet cells (Figure 1D). Consistent with the H&E staining, PAS-positive goblet cells were distributed along the apical side of the culture. Residual secreted mucus also was detected on the apical side by PAS staining.Figure 1

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