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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

Evaluation of transepithelial electrical resistance (TEER) and cellular toxicity in CdCl2-treated human ALI cultures. Cultures were treated from the basolateral side with various concentrations of CdCl2 for 24, 48, or 72 h. The cultures were washed briefly in PBS and TEER measurements (A) were conducted before processing the cultures for cytotoxicity evaluation using the MTS assay (B). Data (N = 3) are presented as means ± standard deviation. *p < 0.05 was considered to be significant compared to the vehicle-treated control.
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Fig2: Evaluation of transepithelial electrical resistance (TEER) and cellular toxicity in CdCl2-treated human ALI cultures. Cultures were treated from the basolateral side with various concentrations of CdCl2 for 24, 48, or 72 h. The cultures were washed briefly in PBS and TEER measurements (A) were conducted before processing the cultures for cytotoxicity evaluation using the MTS assay (B). Data (N = 3) are presented as means ± standard deviation. *p < 0.05 was considered to be significant compared to the vehicle-treated control.

Mentions: TEER measurements were made on CdCl2-treated ALI cultures, followed by the evaluation of cytotoxicity with the MTS assay. Cultures were treated basolaterally with various concentrations of CdCl2 for 24, 48, or 72 h as described in the Methods section. CdCl2 caused dose- and time-dependent decreases in TEER values. Treatment with 100 μM CdCl2 produced an approximate 50% reduction in TEER following both the 24- and 48-h exposures (Figure 2A), and increasing the treatment duration to 72 h further decreased the TEER values close to background levels (i.e., the resistance of an empty culture insert). Treatments with 10 and 30 μM CdCl2 had only minor effects on TEER values, whereas the integrity of the cultures was completely disrupted with a dose of 300 μM CdCl2 at all sampling times.Figure 2


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)

Evaluation of transepithelial electrical resistance (TEER) and cellular toxicity in CdCl2-treated human ALI cultures. Cultures were treated from the basolateral side with various concentrations of CdCl2 for 24, 48, or 72 h. The cultures were washed briefly in PBS and TEER measurements (A) were conducted before processing the cultures for cytotoxicity evaluation using the MTS assay (B). Data (N = 3) are presented as means ± standard deviation. *p < 0.05 was considered to be significant 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

Fig2: Evaluation of transepithelial electrical resistance (TEER) and cellular toxicity in CdCl2-treated human ALI cultures. Cultures were treated from the basolateral side with various concentrations of CdCl2 for 24, 48, or 72 h. The cultures were washed briefly in PBS and TEER measurements (A) were conducted before processing the cultures for cytotoxicity evaluation using the MTS assay (B). Data (N = 3) are presented as means ± standard deviation. *p < 0.05 was considered to be significant compared to the vehicle-treated control.
Mentions: TEER measurements were made on CdCl2-treated ALI cultures, followed by the evaluation of cytotoxicity with the MTS assay. Cultures were treated basolaterally with various concentrations of CdCl2 for 24, 48, or 72 h as described in the Methods section. CdCl2 caused dose- and time-dependent decreases in TEER values. Treatment with 100 μM CdCl2 produced an approximate 50% reduction in TEER following both the 24- and 48-h exposures (Figure 2A), and increasing the treatment duration to 72 h further decreased the TEER values close to background levels (i.e., the resistance of an empty culture insert). Treatments with 10 and 30 μM CdCl2 had only minor effects on TEER values, whereas the integrity of the cultures was completely disrupted with a dose of 300 μM CdCl2 at all sampling times.Figure 2

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