Abstract
The localization and activities of DbpA/ZONAB and YAP transcription factors are in part regulated by the density-dependent assembly of epithelial junctions. DbpA activity and cell proliferation are inhibited by exogenous overexpression of the tight junction (TJ) protein ZO-1, leading to a model whereby ZO-1 acts by sequestering DbpA at the TJ. However, mammary epithelial cells and mouse tissues knock-out for ZO-1 do not show increased proliferation, as predicted by this model. To address this discrepancy, we examined the localization and activity of DbpA and YAP in Madin-Darby canine kidney cells depleted either of ZO-1, or one of the related proteins ZO-2 and ZO-3 (ZO proteins), or all three together. Depletion of only one ZO protein had no effect on DbpA localization and activity, whereas depletion of ZO-1 and ZO-2, which is associated with reduced ZO-3 expression, resulted in increased DbpA localization in the cytoplasm. Only depletion of ZO-2 reduced the nuclear import of YAP. Mammary epithelial (Eph4) cells KO for ZO-1 showed junctional DbpA, demonstrating that ZO-1 is not required to sequester DbpA at junctions. However, further depletion of ZO-2 in Eph4 ZO-1KO cells, which do not express ZO-3, caused decreased junctional localization and expression of DbpA, which were rescued by the proteasome inhibitor MG132. In vitro binding assays showed that full-length ZO-1 does not interact with DbpA. These results show that ZO-2 is implicated in regulating the nuclear shuttling of YAP, whereas ZO proteins redundantly control the junctional retention and stability of DbpA, without affecting its shuttling to the nucleus.
Highlights
ZO-1 overexpression inhibits DbpA/ZONAB overactivation, suggesting that ZO-1 sequesters DbpA at junctions
Our results show that (i) the junctional localization of DbpA is not affected by ZO-1 knockdown or knock-out, in MDCK and Eph4 cells, respectively; (ii) depletion and/or KO of all three ZO proteins is required to observe any effect on DbpA localization and expression; (iii) full-length ZO-1 does not interact with DbpA, and (iv) only ZO-2 regulates the nuclear shuttling of YAP
Simultaneous but Not Individual Depletion of ZO-1, ZO-2, or ZO-3 Is Required to Decrease the Junctional Localization of DbpA in Confluent MDCK Monolayers—To study the role of ZO proteins in regulating the localization and activity of transcription factors DbpA and YAP, we used WT MDCK cells, and distinct clonal lines of MDCK cells stably expressing shRNA, and rescue clones: three clones depleted of ZO-1, a rescue ZO-1 clone (ZO1-R) [26], one clone depleted of ZO-2 (ZO-2KD) [26], one clone depleted of ZO-3 (ZO-3KD), a rescue ZO-3 clone (ZO-3R), and three MDCK clones with a double depletion of ZO-1 and ZO-2 [19]
Summary
ZO-1 overexpression inhibits DbpA/ZONAB overactivation, suggesting that ZO-1 sequesters DbpA at junctions. Evidence from knock-out studies is at variance with the model proposed by Balda and Matter [20], because mammary epithelial cells and mouse tissues lacking ZO-1 do not show altered growth curves, or increased cell proliferation [16, 22, 23] To address these discrepancies, and clarify the role of ZO proteins in the control of DbpA localization, DbpA-dependent gene expression, and cell proliferation, we examined different clonal MDCK lines depleted of ZO-1, ZO-2, or ZO-3, or ZO-1 and ZO-2, and clonal lines of Eph cells, either WT or KO for ZO-1. Our results show that (i) the junctional localization of DbpA is not affected by ZO-1 knockdown or knock-out, in MDCK and Eph cells, respectively; (ii) depletion and/or KO of all three ZO proteins is required to observe any effect on DbpA localization and expression; (iii) full-length ZO-1 does not interact with DbpA, and (iv) only ZO-2 regulates the nuclear shuttling of YAP
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