Related Experiment Videos
Antagonistic regulation of tight junction dynamics by glucocorticoids and transforming growth factor-beta in mouse
1Department of Molecular and Cell Biology, University of California at Berkeley 94720, USA.
Abstract:
The synthetic glucocorticoid, dexamethasone, stimulated the transepithelial electrical resistance and suppressed the DNA synthesis of 31EG4 nontransformed mouse mammary epithelial cells. The addition of transforming growth factor-beta 1 (TGF-beta) to mammary cells simultaneously with or up to 24 h after dexamethasone treatment prevented the steroid induction of transepithelial electrical resistance and stimulated the incorporation of [3H]thymidine. However, the TGF-beta inhibition of tight junction formation did not require de novo DNA synthesis. Confocal microscopy revealed that the organized immunostaining pattern of the tight junction protein, ZO-1, and F-actin at the cell periphery was disrupted by TGF-beta, resulting in disorganized and diffuse staining patterns throughout the cell. Western blot analysis demonstrated that TGF-beta did not alter the protein levels of ZO-1. In contrast to cells not treated or pretreated with steroid for up to 24 h, TGF-beta had no effect on cells pretreated with dexamethasone for 48 h. Transfection of chimeric reporter genes containing promoters responsive to either glucocorticoid or TGF-beta demonstrated that the mutual antagonism of tight junction dynamics by dexamethasone and TGF-beta occurs in the presence of intact signaling pathways. Taken together, our results establish for the first time that glucocorticoids and TGF-beta can antagonistically regulate tight junction formation in a nontransformed mammary cell line.
Insights
Dexamethasone enhances mammary cell barrier function, but transforming growth factor-beta 1 (TGF-beta) disrupts this effect and cell growth. These findings reveal an antagonistic relationship between glucocorticoids and TGF-beta in regulating mammary cell tight junctions.
Area of Science:
- Cell Biology
- Molecular Biology
- Endocrinology
Background:
- Glucocorticoids like dexamethasone influence epithelial cell functions.
- Transforming growth factor-beta 1 (TGF-beta) plays a role in cell growth and differentiation.
- Mammary epithelial cells form tight junctions crucial for barrier function.
Purpose of the Study:
- To investigate the antagonistic effects of dexamethasone and TGF-beta on mammary epithelial cell tight junction formation.
- To elucidate the molecular mechanisms underlying the interaction between glucocorticoid and TGF-beta signaling pathways.
Main Methods:
- Treatment of 31EG4 mouse mammary epithelial cells with dexamethasone and TGF-beta.
- Measurement of transepithelial electrical resistance and DNA synthesis.
- Confocal microscopy to visualize tight junction protein ZO-1 and F-actin.
- Western blot analysis for ZO-1 protein levels.
- Reporter gene assays to assess signaling pathway activity.
Main Results:
- Dexamethasone increased transepithelial electrical resistance and suppressed DNA synthesis.
- TGF-beta reversed dexamethasone-induced transepithelial electrical resistance and stimulated DNA synthesis.
- TGF-beta disrupted the organization of ZO-1 and F-actin at the cell periphery.
- TGF-beta did not affect ZO-1 protein levels, and its inhibitory effect on tight junctions did not require new DNA synthesis.
- TGF-beta's effects were diminished in cells pretreated with dexamethasone for 48 hours.
- Signaling pathways for both hormones remained intact.
Conclusions:
- Glucocorticoids and TGF-beta exhibit an antagonistic interaction in regulating tight junction formation in mammary epithelial cells.
- This antagonism occurs despite intact glucocorticoid and TGF-beta signaling pathways.
- The findings provide novel insights into the complex regulation of epithelial barrier function by hormonal signals.