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Remodeling of Tight Junctions and Enhancement of Barrier Integrity of the CACO-2 Intestinal Epithelial Cell Layer by Micronutrients.

Valenzano MC, DiGuilio K, Mercado J, Teter M, To J, Ferraro B, Mixson B, Manley I, Baker V, Moore BA, Wertheimer J, Mullin JM - PLoS ONE (2015)

Bottom Line: All five of these chemically very diverse micronutrients increased transepithelial electrical resistance (Rt) significantly, but only berberine also improved barrier integrity to the non-electrolyte D-mannitol.The exact effects of the micronutrients on barrier integrity and TJ protein composition were found to be highly dependent on the degree of differentiation of the cell layer at the time it was exposed to the micronutrient.The substratum to which the epithelial layer adheres was also found to regulate the response of the cell layer to the micronutrient.

View Article: PubMed Central - PubMed

Affiliation: Lankenau Institute for Medical Research, Wynnewood, PA, 19096, United States of America.

ABSTRACT
The micronutrients zinc, quercetin, butyrate, indole and berberine were evaluated for their ability to induce remodeling of epithelial tight junctions (TJs) and enhance barrier integrity in the CACO-2 gastrointestinal epithelial cell culture model. All five of these chemically very diverse micronutrients increased transepithelial electrical resistance (Rt) significantly, but only berberine also improved barrier integrity to the non-electrolyte D-mannitol. Increases of Rt as much as 200% of untreated controls were observed. Each of the five micronutrients also induced unique, signature-like changes in TJ protein composition, suggesting multiple pathways (and TJ arrangements) by which TJ barrier function can be enhanced. Decreases in abundance by as much as 90% were observed for claudin-2, and increases of over 300% could be seen for claudins -5 and -7. The exact effects of the micronutrients on barrier integrity and TJ protein composition were found to be highly dependent on the degree of differentiation of the cell layer at the time it was exposed to the micronutrient. The substratum to which the epithelial layer adheres was also found to regulate the response of the cell layer to the micronutrient. The implications of these findings for therapeutically decreasing morbidity in Inflammatory Bowel Disease are discussed.

No MeSH data available.


Related in: MedlinePlus

Differential effect of quercetin treatment on the tight junctional proteins of 7-day vs. 1-day post-confluent CACO-2 cell layers.1-day and 7-day post-confluent CACO-2 cell layers in Falcon 75 cm2 culture flasks were refed with control medium or medium containing 400μM quercetin 48 hrs before harvesting in lysis buffer. Further steps were performed as described in Table 1. Data represents the percentage of band density of the no-quercetin control for that CACO-2 cell layer. Data shown is expressed as the mean ± standard error for an n = 3 cell layers in all cases. * indicates P < 0.05 for 1-day vs. 7-day changes in TJ protein (Student’s t test, two-tailed.
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pone.0133926.g006: Differential effect of quercetin treatment on the tight junctional proteins of 7-day vs. 1-day post-confluent CACO-2 cell layers.1-day and 7-day post-confluent CACO-2 cell layers in Falcon 75 cm2 culture flasks were refed with control medium or medium containing 400μM quercetin 48 hrs before harvesting in lysis buffer. Further steps were performed as described in Table 1. Data represents the percentage of band density of the no-quercetin control for that CACO-2 cell layer. Data shown is expressed as the mean ± standard error for an n = 3 cell layers in all cases. * indicates P < 0.05 for 1-day vs. 7-day changes in TJ protein (Student’s t test, two-tailed.

Mentions: Not only are the TJ complexes changing as the differentiation process proceeds, but the response of the TJ complexes to the various micronutrients seems highly dependent on the state of differentiation at the time that the cell layers are exposed to the micronutrients. As shown in Fig 4, response of the cell layer to supplemental zinc—as evidenced by elevated Rt—is significant for 7- and 21-day cell layers, but not 3-day cell layers, where no effect is observed. This pattern of functional change contrasts sharply with the effects of supplemental zinc on the abundance of the various integral TJ proteins (Fig 5). Zinc markedly and significantly affects the abundance of seven of the eight TJ proteins assayed in 3-day cell layers, but is virtually without effect on the TJ protein abundances of 7-day and 21-day cell layers, where effects are relatively muted. This would appear to be a general phenomenon, and not specific for any single micronutrient. Similar to zinc, quercetin’s effects on TJ protein abundance could be quite different for just-confluent vs. 7-days-post-confluent cell layers (Fig 6). Here, there were significant differences between just-confluent and 7-day-post-confluent cultures regarding claudins -2, -3, -4, -5 and tricellulin. For claudin-2, there was no effect of quercetin in just-confluent cell layers, but a dramatic 150% increase for 7-day cell layers. On the other hand, tricellulin was not affected by quercetin in 7-day cell layers, but its abundance decreased nearly 50% in just-confluent cell layers.


Remodeling of Tight Junctions and Enhancement of Barrier Integrity of the CACO-2 Intestinal Epithelial Cell Layer by Micronutrients.

Valenzano MC, DiGuilio K, Mercado J, Teter M, To J, Ferraro B, Mixson B, Manley I, Baker V, Moore BA, Wertheimer J, Mullin JM - PLoS ONE (2015)

Differential effect of quercetin treatment on the tight junctional proteins of 7-day vs. 1-day post-confluent CACO-2 cell layers.1-day and 7-day post-confluent CACO-2 cell layers in Falcon 75 cm2 culture flasks were refed with control medium or medium containing 400μM quercetin 48 hrs before harvesting in lysis buffer. Further steps were performed as described in Table 1. Data represents the percentage of band density of the no-quercetin control for that CACO-2 cell layer. Data shown is expressed as the mean ± standard error for an n = 3 cell layers in all cases. * indicates P < 0.05 for 1-day vs. 7-day changes in TJ protein (Student’s t test, two-tailed.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4520484&req=5

pone.0133926.g006: Differential effect of quercetin treatment on the tight junctional proteins of 7-day vs. 1-day post-confluent CACO-2 cell layers.1-day and 7-day post-confluent CACO-2 cell layers in Falcon 75 cm2 culture flasks were refed with control medium or medium containing 400μM quercetin 48 hrs before harvesting in lysis buffer. Further steps were performed as described in Table 1. Data represents the percentage of band density of the no-quercetin control for that CACO-2 cell layer. Data shown is expressed as the mean ± standard error for an n = 3 cell layers in all cases. * indicates P < 0.05 for 1-day vs. 7-day changes in TJ protein (Student’s t test, two-tailed.
Mentions: Not only are the TJ complexes changing as the differentiation process proceeds, but the response of the TJ complexes to the various micronutrients seems highly dependent on the state of differentiation at the time that the cell layers are exposed to the micronutrients. As shown in Fig 4, response of the cell layer to supplemental zinc—as evidenced by elevated Rt—is significant for 7- and 21-day cell layers, but not 3-day cell layers, where no effect is observed. This pattern of functional change contrasts sharply with the effects of supplemental zinc on the abundance of the various integral TJ proteins (Fig 5). Zinc markedly and significantly affects the abundance of seven of the eight TJ proteins assayed in 3-day cell layers, but is virtually without effect on the TJ protein abundances of 7-day and 21-day cell layers, where effects are relatively muted. This would appear to be a general phenomenon, and not specific for any single micronutrient. Similar to zinc, quercetin’s effects on TJ protein abundance could be quite different for just-confluent vs. 7-days-post-confluent cell layers (Fig 6). Here, there were significant differences between just-confluent and 7-day-post-confluent cultures regarding claudins -2, -3, -4, -5 and tricellulin. For claudin-2, there was no effect of quercetin in just-confluent cell layers, but a dramatic 150% increase for 7-day cell layers. On the other hand, tricellulin was not affected by quercetin in 7-day cell layers, but its abundance decreased nearly 50% in just-confluent cell layers.

Bottom Line: All five of these chemically very diverse micronutrients increased transepithelial electrical resistance (Rt) significantly, but only berberine also improved barrier integrity to the non-electrolyte D-mannitol.The exact effects of the micronutrients on barrier integrity and TJ protein composition were found to be highly dependent on the degree of differentiation of the cell layer at the time it was exposed to the micronutrient.The substratum to which the epithelial layer adheres was also found to regulate the response of the cell layer to the micronutrient.

View Article: PubMed Central - PubMed

Affiliation: Lankenau Institute for Medical Research, Wynnewood, PA, 19096, United States of America.

ABSTRACT
The micronutrients zinc, quercetin, butyrate, indole and berberine were evaluated for their ability to induce remodeling of epithelial tight junctions (TJs) and enhance barrier integrity in the CACO-2 gastrointestinal epithelial cell culture model. All five of these chemically very diverse micronutrients increased transepithelial electrical resistance (Rt) significantly, but only berberine also improved barrier integrity to the non-electrolyte D-mannitol. Increases of Rt as much as 200% of untreated controls were observed. Each of the five micronutrients also induced unique, signature-like changes in TJ protein composition, suggesting multiple pathways (and TJ arrangements) by which TJ barrier function can be enhanced. Decreases in abundance by as much as 90% were observed for claudin-2, and increases of over 300% could be seen for claudins -5 and -7. The exact effects of the micronutrients on barrier integrity and TJ protein composition were found to be highly dependent on the degree of differentiation of the cell layer at the time it was exposed to the micronutrient. The substratum to which the epithelial layer adheres was also found to regulate the response of the cell layer to the micronutrient. The implications of these findings for therapeutically decreasing morbidity in Inflammatory Bowel Disease are discussed.

No MeSH data available.


Related in: MedlinePlus