Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Jul 30;10(7):e0133926.
doi: 10.1371/journal.pone.0133926. eCollection 2015.

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

Mary Carmen Valenzano  1 , Katherine DiGuilio  1 , Joanna Mercado  1 , Mimi Teter  2 , Julie To  2 , Brendan Ferraro  2 , Brittany Mixson  1 , Isabel Manley  2 , Valerissa Baker  2 , Beverley A Moore  3 , Joshua Wertheimer  3 , James M Mullin  4
Affiliations

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

Mary Carmen Valenzano et al. PLoS One. .

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.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. The effect of six micronutrients on CACO-2 transepithelial electrical resistance.
1A: 7-Day post-confluent CACO-2 cell layers on Millipore PCF filters were refed in control medium (apical and basal-lateral compartments) or medium containing 50μM, 100μM, or 200μM berberine chloride 12–17 hrs prior to electrical measurements. Data shown represents the mean ± standard error of 9 cell layers per condition (3 experiments, 3 cell layers per experiment). 1B: 7-Day post-confluent CACO-2 cell layers on Millipore PCF filters were refed, as above, in control medium or medium containing 1μM, 10μM, or 100μM Nicotine 48 hrs prior to electrical measurements. Data shown represents the mean ± standard error of 6 cell layers per condition (2 experiments, 3 cell layers per experiment). 1C: 4-Day post-confluent CACO-2 cell layers on Millipore PCF filters were refed, as in A, in control medium or medium containing 0.5mM, 2.0mM, or 5mM sodium butyrate 72 hrs prior to electrical measurements. Data shown represents the mean ± standard error of 6 cell layers per condition (2 experiments, 3 cell layers per experiment). 1D: 7-Day post-confluent CACO-2 cell layers on Millipore PCF filters were refed, as in A, in control medium or medium containing 100μM, 200μM, or 400μM quercetin 48hrs prior to electrical measurements. Data shown represents the mean ± standard error of 6 cell layers per condition (2 experiments, 3 cell layers per experiment). 1E: 7-Day post-confluent CACO-2 cell layers on Millipore PCF filters were refed, as in A, in control medium or medium containing 0.5mM, 1.0mM, or 2.0mM indole 48hrs prior to electrical measurements. Data shown represents the mean ± standard error of 9 cell layers per condition (3 experiments, 3 cell layers per experiment). 1F: 7-Day post-confluent CACO-2 cell layers on Millipore PCF filters were refed, as in A, in control medium or medium containing 50μM, 100μM, or 150μM zinc sulfate 48hrs prior to electrical measurements. Data shown represents the mean ± standard error of 12 cell layers for both the control and 100μM conditions, and 4 cell layers for both the 50μM and 150μM conditions. In all cases, data represents the percent of control resistance normalized for each experiment. * indicates P
Fig 2
Fig 2. The effect of six micronutrients on CACO-2 transepithelial flux of 14C-D-Mannitol.
In all cases, after electrical measurements, the same CACO-2 cell layers represented in Fig 1 were used to perform radiotracer flux studies with 0.1mM, 0.20 μCi/ml 14C-D-mannitol, as described in Materials and Methods. Data represents the percent of control flux rate normalized for each experiment, and is expressed as the mean ± standard error for the total number of cell layers per condition, as detailed in Fig 1. * indicates P < 0.05, ** indicates P < 0.01 and *** indicates P < 0.001 (one-way ANOVA followed by Dunnett’s post hoc testing versus control).
Fig 3
Fig 3. Change of tight junctional proteins as a function of state of differentiation of the CACO-2 cell layer.
The relative changes in the abundance of eight tight junctional proteins occurring as a function of days post confluence of the cell layer. 3-day, 7-day, and 21-day post-confluent CACO-2 cell layers in Falcon 75 cm2 culture flasks, having been refed with control medium at confluence and every 2–3 days thereafter with control medium, were harvested in lysis buffer. Further steps were performed as described in Table 1. Data represents the percent of 3-day cell layers, and is expressed as the mean ± standard error for an n = 3 cell layers in all cases. NS indicates non significance. * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001 (one-way ANOVA followed by Dunnett’s post hoc testing versus day 3).
Fig 4
Fig 4. Effect of zinc on CACO-2 transepithelial electrical resistance as a function of the differentiation state of the CACO-2 cell layer.
3-day, 7-day, and 21-day CACO-2 cell layers were treated on the apical and basal-lateral sides with 100μM zinc for 48 hrs before electrical measurements. Data represents the percentage of normalized control resistance (2 experiments, 4 cell layers per condition per experiment) as a function of zinc treatment. Data shown is expressed as the mean ± standard error of 8 cell layers per condition. ** indicates P
Fig 5
Fig 5. Effect of zinc on CACO-2 tight junctional proteins as a function of the differentiation state of the CACO-2 cell layer.
3-day, 7-day, and 21-day post-confluent CACO-2 cell layers in Falcon 75 cm2 culture flasks were refed with control medium or medium containing 100μM Zinc 48 hrs before harvesting in lysis buffer. Further steps were performed as described in Table 1. Data represents the percent of the zinc condition relative to the normalized control for each age cell layer. Data shown is expressed as the mean ± standard error for an n = 4 cell layers in all cases. NS indicates non significance. * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001 (Student’s t test, two-tailed).
Fig 6
Fig 6. 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.
Fig 7
Fig 7. The effect of the cell layer substratum on CACO-2 epithelial barrier’s response to zinc CACO-2 cell layers were seeded at identical densities on Millicell PCF and Millicell HA units as described in Materials and Methods.
After 7 days, cell layers were refed in apical and basal-lateral compartments with control medium or medium containing 100μM zinc, 48 hrs before electrical measurements. Data represents the percentage of normalized control resistance, normalized control short circuit current, and normalized control flux rate (each relative to proper control; 2 experiments, 4 cell layers per condition). Data shown is expressed as the mean ± standard error of 8 cell layers per condition. P values (Student’s t test, two-tailed) are indicated for statistical comparisons of the various conditions.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Mullin JM, Agostino N, Rendon-Huerta E, Thornton JJ (2005) Keynote review: epithelial and endothelial barriers in human disease. Drug Discov Today 10: 395–408. - PubMed
    1. Sawada N (2013) Tight junction-related human diseases. Pathol Int 63: 1–12. 10.1111/pin.12021 - DOI - PMC - PubMed
    1. Edelblum KL, Turner JR (2009) The tight junction in inflammatory disease: communication breakdown. Curr Opin Pharmacol 9: 715–720. 10.1016/j.coph.2009.06.022 - DOI - PMC - PubMed
    1. Schulzke JD, Ploeger S, Amasheh M, Fromm A, Zeissig S, Troeger H., et al. (2009) Epithelial tight junctions in intestinal inflammation. Ann N Y Acad Sci. 1165: 294–300 10.1111/j.1749-6632.2009.04062.x - DOI - PubMed
    1. Hering NA, Schulzke JD (2009) Therapeutic options to modulate barrier defects in inflammatory bowel disease. Dig Dis 27: 450–454. 10.1159/000233283 - DOI - PubMed

Publication types